WO2003064655A1 - Sugar chain synthases - Google Patents

Sugar chain synthases Download PDF

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Publication number
WO2003064655A1
WO2003064655A1 PCT/JP2003/000883 JP0300883W WO03064655A1 WO 2003064655 A1 WO2003064655 A1 WO 2003064655A1 JP 0300883 W JP0300883 W JP 0300883W WO 03064655 A1 WO03064655 A1 WO 03064655A1
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Prior art keywords
amino acid
sequence
sialyltransferase
acid sequence
seq
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PCT/JP2003/000883
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French (fr)
Japanese (ja)
Inventor
Shou Takashima
Masafumi Tsujimoto
Shuichi Tsuji
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Riken
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Priority to US10/501,930 priority Critical patent/US20060057696A1/en
Priority to JP2003564247A priority patent/JP4429018B2/en
Publication of WO2003064655A1 publication Critical patent/WO2003064655A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)

Definitions

  • the present invention relates to a sugar chain synthase and a DNA encoding the enzyme. More specifically, the present invention relates to the sialic acid moiety of a sugar chain having a Sia o; 2,3 (6) Gal (Sia: sialic acid, Gal: galactose) structure at the end of an O-type sugar chain such as mucin.
  • An enzyme (0-glycan ⁇ 2,8-sialyltransferase, ST8Sia VI) that efficiently transfers sialic acid in a binding mode of 2,8, and DNA encoding the enzyme; and sugar chains such as oligosaccharides of, Gal 1 to end, 4GlcNAc (Gal: galactose, GlcNAc: - Asechiruguru Kosamin) 2 alpha to galactose moiety of a sugar chain having a structure, 6 enzymes that efficiently transfers sialic acid binding modes (ST6Gal II ) And DNA encoding the enzyme.
  • sugar chains such as oligosaccharides of, Gal 1 to end, 4GlcNAc (Gal: galactose, GlcNAc: - Asechiruguru Kosamin) 2 alpha to galactose moiety of a sugar chain having a structure
  • 6 enzymes that efficiently transfers sialic acid binding modes (ST6Gal II ) And DNA
  • the O-glycan ct 2,8-sialyltransferase and] 3-galactoside ⁇ 2,6-sialyltransferase of the present invention can suppress cancer metastasis, prevent virus infection, suppress inflammatory response, and activate nerve cells. It is useful as a drug having the same, or as a reagent for increasing a physiological action by adding sialic acid to a sugar chain, or as an enzyme inhibitor.
  • Sialic acid is a substance that controls important physiological actions such as cell-cell communication, cell-substrate interaction, and cell adhesion. It is known that sialic acid-containing sugar chains specific to the process of development and differentiation or organ-specific are present. Sialic acid exists at the terminal position of the sugar chain portion of glycoproteins and glycolipids, and the introduction of sialic acid into these sites is performed by enzymatic transfer from CMP-Sia.
  • sialyltransferases glycosyltransferases
  • sialyltransferases glycosyltransferases.
  • sialyltransferases glycosyltransferases in mammals, It is roughly classified into four families according to the mode of transfer of lactic acid (Tsuji, S. (1996) J. Biochem. 120, 1-13).
  • ⁇ 2,3-sialyltransferase ST3Gal-family
  • ST3Gal-family which transfers sialic acid to galactose in the ⁇ 2,3 binding mode, transfers sialic acid to galatatoose in the ⁇ 2,6 bonding mode.
  • ST8Sia I is a GD3 synthase of gandarioside
  • ST8Sia V is an enzyme that synthesizes GDlc, GTla, GQlb, GT3, etc. of gandarioside
  • ST8Sia II and IV are enzymes that synthesize polysialic acid on the N-type sugar chain of nerve cell adhesion molecule (NCAM).
  • ST8Sia III is an enzyme which transfers sialic acid to Sia a 2,3Gal ⁇ l, 4GlcNAc structure found in N-glycans and glycolipids glycoproteins.
  • ST6Gal I has activity on Gal ⁇ 1,4GlcNAc structures such as glycoproteins, oligosaccharides and gandariosides, but has the activity of Gal ⁇ 1,4GlcNAc as well as ratatose (Gal / 31, It is an enzyme with a wide substrate specificity that can be used as a substrate even with 4Glc) or, in some cases, Gal / 31 / 3GlcNAc structure.
  • the wide substrate specificity means that, for example, when synthesizing functional oligosaccharides using ST6Gal I, if impurities are mixed in the raw materials, they may also become substrates and generate by-products. Sex is considered.
  • a first object of the present invention is to provide a novel O-glycan ⁇ 2,8-sialyltransferase having high activity on type I sugar chains.
  • the present invention provides a method for cloning a cDNA encoding 0-glycan ct 2,8-sialyltransferase, a DNA sequence encoding the O-glycan ⁇ 2,8-sialyltransferase, and an amino acid sequence of the enzyme.
  • the purpose is to provide.
  • Another object of the present invention is to express a portion of the structure of the above-mentioned O-glycan ⁇ 2,8-sialyltransferase involved in activity as a protein in a large amount.
  • a second object of the present invention is to solve the problem of wide substrate specificity, and to provide a novel substrate specificity that shows higher selectivity for the Gal ⁇ 1,4GlcNAc structure on oligosaccharides.
  • An object of the present invention is to provide a galactoside ⁇ 2,6-sialyltransferase and a DNA encoding the enzyme.
  • the present inventors have made intensive efforts to solve the above problems, screened mouse brain and heart cDNA libraries, and performed O-glycan ct 2 We succeeded in cloning cDNA encoding 8-, 8-sialyltransferase. Furthermore, the present inventor has used the amino acid sequence of the human sialyltransferase ST6Gal I to encode a novel sialyltransferase showing homology thereto, and cloned the clone with the expressed sequence tag (dbEST). Search was performed using a ⁇ database to obtain EST clones of GenBank TM accession Nos. BE613250, BE612797, and BF038052.
  • ⁇ -glycan ⁇ 2, 8-sialyltransferase which has the following substrate specificity and substrate selectivity.
  • Substrate specificity using a sugar having a terminal Sia a 2, 3 (6) Gal (where Sia represents sialic acid and Gal represents galactose) substrate;
  • Substrate selectivity Incorporates sialic acid into O-glycans preferentially over glycolipids and N-glycans:
  • the present invention provides an O-glycan a 2,8-sialyltransferase having any one of the following amino acid sequences.
  • O-glycan a 2, 8-sialic acid having an amino acid sequence having deletion, substitution and / or addition of one to several amino acids in the amino acid sequence described in SEQ ID NO: 1 or 3 in the sequence listing.
  • an O-glycan ⁇ 2,8-sialyltransferase gene encoding the amino acid sequence of the above-described O-glycan 2,8-sialyltransferase of the present invention.
  • the present invention provides an O-glycan «2, 8- sialyltransferase having any one of the following nucleotide sequences.
  • nucleotide sequence specified by nucleotide numbers 77 to 127 in the nucleotide sequence of SEQ ID NO: 2 in the sequence listing;
  • a recombinant vector (preferably, an expression vector) containing the ⁇ -glycan ⁇ 2,8-sialyltransferase gene of the present invention described above; And a method for producing the enzyme of the present invention, which comprises culturing the transformant and collecting the enzyme of the present invention from the culture.
  • a protein comprising -glycan ⁇ 2, 8-sialyltransferase active domain.
  • amino acid sequence consisting of amino acid numbers 26-398 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing;
  • amino acid sequence comprising amino acid numbers 68 to 398 of the amino acid sequence described in SEQ ID NO: 3 in the sequence listing, the amino acid sequence having one to several amino acid deletions, substitutions, and / or additions
  • amino acid sequence having the activity of catalyzing the transfer of ⁇ -glycana 2,8_sialyl acid
  • an extracellular secretory protein comprising a polypeptide portion, which is an active domain of the O-glycan ⁇ 2,8-sialyltransferase of the present invention, and a signal peptide And a protein having an activity of catalyzing 0-glycan ⁇ 2,8-sialyltransfer is provided.
  • a recombinant vector (preferably, an expression vector) containing a gene encoding the above-described extracellular secretory protein of the present invention; a transformant transformed with the above-described recombinant vector; And a protein of the present invention, which comprises culturing the above-mentioned transformant and collecting the enzyme of the present invention from the culture.
  • a quality manufacturing method is provided.
  • 3-galactoside 2,6-sialyltransferase which has the following action and substrate specificity.
  • [Galactose at the terminal] 31,4 N-Acetyldarcosamine structure is used as a substrate, and lactose and a sugar chain having a galactose / 31,3N-acetyldarcosamine structure at the end are not used as substrates. .
  • a monogalactoside ⁇ 2,6-sialyltransferase gene encoding the amino acid sequence of the above-mentioned J3-galactoside 2,6-sialyltransferase of the present invention.
  • an i3-galactoside ⁇ 2,6-sialyltransferase gene having any one of the following nucleotide sequences.
  • nucleotide sequence encoding a protein having an activity of catalyzing the transfer of 3-galactoside ⁇ 2,6-sialyl acid (3) a nucleotide sequence identified by nucleotide number 3 to nucleotide 157 in the nucleotide sequence of SEQ ID NO: 8 in the sequence listing; or
  • a nucleotide sequence encoding a protein having an activity to catalyze the transfer of / 3-galactoside ⁇ 2,6-sialyl acid having the following nucleotide sequence:
  • a recombinant vector comprising the / 3_galactoside ⁇ 2,6-sialyltransferase gene of the present invention.
  • the recombinant vector of the present invention is preferably an expression vector.
  • a method for producing the enzyme of the present invention which comprises culturing the transformant of the present invention and collecting the enzyme of the present invention from the culture.
  • a protein comprising a 3-galactoside ⁇ 2,6-sialyltransferase active domain having any one of the following amino acid sequences:
  • amino acid sequence consisting of amino acid numbers 33 to 529 of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing;
  • amino acid sequence having 1 to several amino acid deletions, substitutions and ⁇ or additions in the amino acid sequence consisting of amino acids 33 to 529 in the amino acid sequence described in SEQ ID NO: 5 in the sequence listing
  • amino acid sequence having an amino acid sequence consisting of amino acids Nos. 31 to 524 of the amino acid sequence described in SEQ ID NO: 7 in the sequence listing, which has one to several amino acid deletions, substitutions and no or additions Catalyzes the transfer of / 3-galactoside ⁇ 2,6-sialyl acid Amino acid sequence having the following activities:
  • an extracellular secretory type comprising a polypeptide portion, which is an active domain of the 3-galactoside ⁇ 2,6-sialyltransferase of the present invention, and a signal peptide.
  • a protein which has an activity of catalyzing a / 3-galactoside ⁇ 2,6-sialyltransferase.
  • a recombinant vector comprising the above-described gene of the present invention.
  • the recombinant vector of the present invention is preferably an expression vector.
  • a method for producing the protein of the present invention which comprises culturing the transformant of the present invention and collecting the protein of the present invention from the culture.
  • FIG. 1 shows the nucleotide sequence and the predicted amino acid sequence of ST8Sia VI cDNA from mouse mouse.
  • the transmembrane domain is underlined, the sialyl motif L is shown as a double line, and the sialyl motif S is shown as a dashed line. Histidine and daltamic acid conserved in the sialyl motif VS are boxed. Asparagine to which type-glycans are expected to bind is underlined.
  • A mouse ST8Sia VI.
  • B human ST8Sia VI.
  • FIG. 2 shows a comparison of the amino acid sequences.
  • A shows a comparison of the amino acid sequences of mouse sialyltransferases ST8Sia I, ST8Sia V, and ST8Sia VI. Amino acids conserved between each sialyltransferase are boxed. The Cyaryl motif L is shown by a double line, and the Cyaryl motif S is shown by a broken line. You. Histidine and glutamic acid conserved in the sialyl motif VS are marked with an asterisk.
  • FIG. 3 shows an analysis of the binding specificity.
  • FIG. 4 shows the result of treating Fetuin having [ 14 C]-NeuAc incorporated by ST8Sia III or ST8Sia VI with -glycanase.
  • Fetuin incorporating [ 14 C] -NeuAc was treated with -glycanase, analyzed by SDS-PAGE, and visualized with a BAS2000 radio image analyzer.
  • FIG. 5 shows the effect of overexpressing mouse ST8Sia VI full-length cDNA in C0S-7 cells.
  • A shows the results of T and C immunostaining performed using anti-euAca2, 8NeuAca2, and 3Gal antibody S2-566.
  • Lane 1 GD3 standard (0.5 g); Lane 2, GQlb standard (0.5 ⁇ g); Lane 3, acidic glycolipid fraction extracted from control COS-7 cells (30 mg); Lane 4, mouse C0S-7 transfected with full-length ST8Sia VI expression vector pRc / CMV-ST8Sia VI Acid glycolipid fraction extracted from cells (30 mg).
  • B is a microsomal fraction prepared from COS- 7 cells transfected with C0S-7 cells or P Rc / CMV- ST8Sia VI, was subjected to SDS- PAGE (45 g / lane), transferred to PVDF membrane 13 shows the results of Western blotting using the S2-566 antibody.
  • FIG. 6 shows the expression modes of ST8Sia VI gene in mouse and human.
  • A shows the results of Northern analysis of the expression mode of the mouse ST8Sia VI gene using poly (A) + RNA (about 2 ⁇ g / lane) prepared from various mouse organs.
  • FIG. B shows the result of analyzing the expression mode of the human ST8Sia VI gene by PCR using the Multiple Tissue cDNA Panel (Clontech).
  • human ST8Sia VI-specific primers 5′-CCAGTGTCCCAGCCTTTTGT-3 ′ (corresponding to base numbers 608-627 in FIG. 1B) (SEQ ID NO: 17) and 5′-TGAGTGGGGAAGCTTTGGTC-3 ′ (base in FIG. 1B) No. 1407-1426) (SEQ ID NO: 18) was used (the size of the PCR amplified fragment was 819 bp).
  • FIG. 7 shows the nucleotide sequence of human ST6Gal II cDNA, the predicted amino acid sequence, and its hydrophobicity distribution map.
  • A shows the nucleotide sequence of human ST6Gal II cDNA and the predicted amino acid sequence.
  • the transmembrane domain is underlined, the sialyl motif L is shown as a double line, and the sialyl motif S is shown as a dashed line. Histidine and glutamic acid conserved in the sialyl motif VS are boxed. Asparagine to which type-glycans are expected to bind is underlined.
  • FIG. 8 shows the nucleotide sequence and predicted amino acid sequence of mouse ST6Gal II cDNA, and the hydrophobic distribution map thereof.
  • A shows the nucleotide sequence of mouse ST6Gal II cDNA and the predicted amino acid sequence. Transmembrane domains are underlined, sialyl motifs L are double lines, and sialyl motifs S are dashed. Histidine and glutamic acid conserved in the sialyl motif VS are boxed. Asparagine to which type-glycans are expected to bind is underlined.
  • FIG. 9 shows a comparison of the amino acid sequences.
  • A shows a comparison of the amino acid sequences of human ST6Gal I and ST6Gal II. Amino acids conserved between both sialyltransferases are boxed. The sialyl motif L is shown as a double line, and the sialyl motif S is shown as a dashed line. Histidine and glutamic acid conserved in the sialyl motif VS are marked with an asterisk.
  • FIG. 11 shows an analysis of binding specificity.
  • FIG. 12 shows the analysis of the expression patterns of the human ST6Gal I, ST6Gal II and mouse ST6Gal II genes. Expression patterns of both genes were analyzed by PCR using human ST6Gal I and ST6Gal II specific primers and a multiple tissue cDNA panel (Clontech) of human tissue ( ⁇ ) or human tumor cells ( ⁇ ). PCR was performed at 94 ° C for 1 minute, 50 ° C for 1 minute, 72 ° C for 1 minute and 30 seconds, and 25 cycles for Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene and 40 cycles for human ST6Gal I and ST6Gal II genes The reaction products were analyzed by agarose gel electrophoresis. Sk.
  • Panel C shows the results of PCR analysis of the expression pattern of mouse ST6Gal II using mouse ST6Gal II-specific primers and a mouse tissue Multiple tissue cDNA panel (Clontech). BEST MODE FOR CARRYING OUT THE INVENTION
  • the O-glycan ⁇ 2, 8-sialyltransferase of the present invention is characterized by having the following substrate specificity and substrate selectivity.
  • Substrate specificity a sugar having a structure of Sia a 2, 3 (6) Gal (where Sia represents sialic acid and Gal represents galactose) at the terminal is used as a substrate;
  • Substrate selectivity Incorporates sialic acid into O-glycans preferentially over glycolipids and N-glycans: The above-described substrate specificity and substrate selectivity are properties demonstrated for the mouse and human O-glycan ⁇ 2,8-sialyltransferase obtained in the examples described in the present specification.
  • the origin of the O-glycan o; 2,8-sialyltransferase of the present invention is not limited to those derived from mice and humans, and the same type of ⁇ -glycan ⁇ 2, 8-sialoletransferase It is easily understood by those skilled in the art that is present in other mammalian tissues, and that the O-glycan ⁇ 2,8-sialyltransferase has a high degree of homology to each other.
  • Such O-glycan ⁇ 2,8-sialyltransferase is characterized by having the above-described substrate specificity and substrate selectivity, and all belong to the scope of the present invention.
  • enzymes include natural enzymes derived from mammalian tissues and mutants thereof, and ⁇ ⁇ ⁇ -glycan ⁇ 2,8-sialyltransfer such as those prepared in the examples below, and are produced by gene recombination techniques. And extracellular secretory proteins, which are all included in the scope of the present invention.
  • An example of the 0-glycan ⁇ 2,8-sialyltransferase of the present invention includes ⁇ -glycan ⁇ 2,8-sialyltransferase having any of the following amino acid sequences.
  • the active domain of the O-glycan ⁇ 2,8-sialyltransferase of the present invention or the O-glycan ct 2,8-sialyltransferase obtained by modifying or modifying a part of the amino acid sequence thereof
  • all active proteins are included in the scope of the present invention.
  • Preferred examples of such an active domain are specified by 26 to 398 of the amino acid sequence described in SEQ ID NO: 1 of the Sequence Listing or 68 to 398 of the amino acid sequence described in SEQ ID NO: 3 in the Sequence Listing.
  • O-glycan ⁇ 2,8-sialyltransferase activity domain are also described in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing.
  • sequence of about 26 to 100 in the amino acid sequence described above is considered to be not necessarily essential for the activity since it is a region called a stem. Therefore, the region from 101 to 398 of the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing was used as the active domain of O-glycan a 2,8-sialyltransferase. Is also good.
  • a protein comprising an O-glycan ⁇ 2,8-sialyltransferase active domain having any of the following amino acid sequences.
  • amino acid sequence consisting of amino acid numbers 26 to 398 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing;
  • amino acid sequence having deletion, substitution, and ⁇ or addition of one to several amino acids in the amino acid sequence consisting of amino acid numbers 68 to 398 of the amino acid sequence described in SEQ ID NO: 3 in the sequence listing
  • the / 3-galactoside ⁇ 2,6-sialyltransferase of the present invention is characterized by having the following action and substrate specificity.
  • Sialic acid is transferred to the galactose part of the sugar chain having a galactose / 31,4-percetyldarcosamine structure at the terminal by ⁇ 2,6 binding.
  • Lactose and a sugar chain having a galactose J31,3N-acetyldarcosamine structure at the terminal using a sugar chain having a 31,4 1-acetylacetylcosamine structure as a substrate Is not used as a substrate.
  • the actions and substrate specificities described above are properties demonstrated for the human and mouse-derived] 3-galactoside ⁇ 2,6-sialyltransferase obtained in the examples described in the present specification. .
  • the origin of the -galactoside ⁇ 2,6-sialyltransferase of the present invention is not limited to that derived from human or mouse, and the same type of] -galactoside ⁇ 2,6-sialyltransferase is used. It will be readily appreciated by those skilled in the art that the [3-galactoside ⁇ 2,6-sialyltransferases present in other mammalian tissues and have a high degree of homology to one another.
  • Such a] 3-galactoside ⁇ 2,6-sialyltransferase is characterized by having the above-mentioned action and substrate specificity, and all belong to the scope of the present invention.
  • enzymes include natural enzymes derived from mammalian tissues and mutants thereof, and extracellular secretory proteins produced by gene recombination technology that catalyze the transfer of ⁇ -galatatoside ⁇ 2,6-sialyl acid. However, these are all included in the scope of the present invention.
  • An example of the [3-galactoside ⁇ 2,6-sialyltransferase of the present invention] is a / 3-galactoside ⁇ 2,6-sialyltransferase having any of the following amino acid sequences.
  • the [3-galactoside ⁇ 2,6-sialyltransferase of the present invention] is obtained by modifying or modifying an active domain thereof or a part of the amino acid sequence thereof / 3-galactoside ct2,6- It should be understood that all proteins having sialyltransferase activity are included in the scope of the present invention. Preferred examples of such an active domain include those specified in 33 to 529 of the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing.] 3 Activity of monogalactoside "2,6-sialyltransferase Domain You.
  • sequence of about 31 to 200 of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing is a region called a stem, it is considered that the sequence is not necessarily essential for activity. Therefore, the region from 201 to 5229 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing may be used as the active domain of) 3-galactoside ⁇ 2,6-sialyltransferase.
  • an active domain of ⁇ -galatatoside ⁇ 2,6-sialyltransferase identified by 31 to 524 of the amino acid sequence described in SEQ ID NO: 7 in the sequence listing may be used. Can be mentioned.
  • the sequence from 3 :! to around 200 in the amino acid sequence described in SEQ ID NO: 7 in the sequence listing is a region called a stem, it is considered that the sequence is not necessarily essential for the activity. Therefore, the region of 201 to 524 of the amino acid sequence described in SEQ ID NO: 7 in the sequence listing may be used as the active domain of / 3_galactoside ⁇ 2,6-sialyltransferase.
  • a protein comprising any of the following amino acid sequences: 3_galactoside ⁇ 2,6-sialyltransferase active domain.
  • a protein comprising a / 3-galactoside ⁇ 2,6-sialyltransferase active domain having any one of the following amino acid sequences.
  • amino acid sequence consisting of amino acid numbers 33 to 529 of the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing;
  • ⁇ 1 to several '' in the ⁇ amino acid sequence having deletion, substitution and Z or addition of 1 to several amino acids '' referred to herein is not particularly limited, for example, 1 to 20 It preferably means about 1 to 10, more preferably about 1 to 7, even more preferably about 1 to 5, particularly preferably about 1 to 3.
  • the method for obtaining the enzyme or protein of the present invention is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by a genetic recombination technique.
  • Methods for isolating cDNA encoding monogalactoside ⁇ 2,6-sialyltransferase are described in detail in the Examples below.
  • the method for isolating the cDNA encoding the O-glycan ⁇ 2,8-sialyltransferase or] 3-galatatoside ⁇ 2,6-sialyltransferase of the present invention is limited to these methods.
  • those skilled in the art can easily isolate the desired cDNA by appropriately modifying or changing this method while referring to the method described in the following Examples.
  • DNA can be obtained.
  • the enzyme of the present invention can be produced by introducing this DNA into an appropriate expression system. Departure The expression in the current system will be described later in this specification.
  • the present invention relates to an extracellular secretory protein comprising a polypeptide portion which is an active domain of 0-glycan ⁇ 2,8-sialyltransferase or -galactoside ⁇ 2,6-sialyltransferase of the present invention and a signal peptide.
  • a protein having an activity of catalyzing O-glycan ⁇ 2,8-sialyltransfer or ⁇ -galactoside ⁇ 2,6-sialyltransfer is also included in the present invention.
  • the 0-glycan ⁇ 2,8-sialyltransferase of the present invention] -galactoside ⁇ 2,6-sialyltransferase may remain in the cell after expression and may not be secreted out of the cell. In addition, when the intracellular concentration exceeds a certain level, the expression level of the enzyme may decrease.
  • Such proteins include the 0-glycan ⁇ 2,8_sialyltransferase or the O-glycan ⁇ 2,8 involved in the activity of / 3-galactoside ⁇ 2,6-sialyltransferase of the present invention.
  • 0-glycan ⁇ 2 is an extracellular secretory protein containing the polypeptide portion and the signal peptide, which are the active domain of -sialyltransferase or / 3-galactoside 2,6-sialyltransferase. , 8-sialyltransfer or j3_galactoside ⁇ 2,6 -sialyltransfer.
  • a signal peptide of mouse immunoglobulin IgM and a fusion protein with protein A are preferred embodiments of the secretory protein of the present invention.
  • sialyltransferases that have been clawed have a domain structure similar to that of other dalycosyltransferases. That, NH 2 terminal short cytoplasmic Nakao unit, hydrophobic signal anchor domain, a stem (stem) regions with proteolytic susceptibility, has a large active domain of ⁇ Pi C00H- end (Paulson, JC and Col ley, KJ , J. Biol. Chew., 264, 17615-17618, 1989).
  • the O-glycan a 2,8-sialyltransferase or the 3-galactoside a 2,6-sialyltransferase of the present invention
  • a hydrophobic distribution prepared according to the method of Kite and Doolittle (RF, J. Mol. Biol., 157, 105-132, 1982) was used. Figures are available.
  • RF J. Mol. Biol., 157, 105-132, 1982
  • Figures are available.
  • a recombinant plasmid into which various types of fragments are introduced can be used.
  • One example of such a method is described in detail in, for example, the specification of PCT / JP94 / 02182, but the method of confirming the location of the transmembrane domain and estimating the active domain portion is limited to this method. None.
  • O-glycan ⁇ 2, 8-sialyltransferase or] 3-galactoside ⁇ 2,6-sialyltransferase The production of extracellular secretory proteins containing the polypeptide part of the active domain and the signal peptide
  • the immunoglobulin signal peptide sequence as the signal peptide, it corresponds to the active domain of O-glycan 2,8-sialyltransferase or] 3-galactoside ⁇ 2,6-sialyltransferase
  • a sequence to be fused to the signal peptide in frame for example, Joblin's method (Jobling, S.A.
  • a fusion protein with mouse A immunoglobulin IgM signal peptide or protein A may be produced.
  • the type of signal peptide, the method of binding the signal peptide to the active domain, or the method of solubilization are not limited to the above methods, and those skilled in the art may use O-glycan ⁇ 2, 8-sialyltransferase.
  • polypeptide moiety that is the active domain of 3-galactoside ⁇ 2,6-sialyltransferase can be selected as appropriate, and they can be bound to any available signal peptide by an appropriate method. As a result, an extracellular secretory protein can be produced.
  • a gene encoding the amino acid sequence of 0-glycan ⁇ 2,8-sialyltransferase of the present invention and a gene encoding 3_galactoside ⁇ 2,6-sialyltransferase
  • a gene encoding a amino acid sequence is provided.
  • genes encoding the amino acid sequence of the O-glycan ⁇ 2,8_sialyltransferase of the present invention include a gene having any one of the following nucleotide sequences.
  • nucleotide sequence identified by nucleotide numbers 77 to 127 in the nucleotide sequence of SEQ ID NO: 2 in the sequence listing;
  • nucleotide sequence set forth in SEQ ID NO: 4 in the nucleotide sequence listed in SEQ ID NO: 4 there is a deletion, substitution, and / or addition of one to several nucleotides in the nucleotide sequence specified by nucleotide numbers 9 to 1285
  • genes encoding the amino acid sequence of the 3-galactoside ⁇ 2,6-sialyltransferase of the present invention include a gene having any one of the following nucleotide sequences.
  • nucleotide sequence specified by nucleotide numbers 176 to 176 in the nucleotide sequence of SEQ ID NO: 6 in the sequence listing;
  • ⁇ 1 to several '' in the ⁇ base sequence having 1 to several bases of deletion, substitution and Z or addition '' referred to herein is not particularly limited, for example, 1 to 60, Preferably about 1 to 30, more preferably about 1 to 20, more preferably about 1 to 10, more preferably about 1 to 5, particularly preferably about 1 to 3.
  • a protein comprising an active domain of the O-glycan ⁇ 2,8-sialyltransferase or j3-galactoside a2,6-sialyltransferase of the present invention, and a polypeptide moiety which is the active domain
  • An extracellular secretory protein containing a protein and a signal peptide, which encodes a protein having an activity of catalyzing O-glycan ⁇ 2,8-sialyltransfer or / 3-galactoside ⁇ 2,6-sialyltransferase Genes belonging to the present invention also belong to the scope of the present invention.
  • the method for obtaining the gene of the present invention is as described above.
  • a method for introducing a desired mutation into a predetermined nucleic acid sequence is known to those skilled in the art.
  • a DNA having a mutation is constructed by appropriately using known techniques such as site-directed mutagenesis, PCR using a degenerate oligonucleotide, exposure of a cell containing nucleic acid to a mutagen or radiation. can do.
  • known techniques such as site-directed mutagenesis, PCR using a degenerate oligonucleotide, exposure of a cell containing nucleic acid to a mutagen or radiation. can do.
  • Such known techniques the example, Molecular Cloning:.. A laboratory annual, 2 nd Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, and Current Protocols in Molecular Biology, Supplement 1 ⁇ 38 , John Wiley & Sons (1987-1997).
  • the gene of the present invention can be used by inserting it into an appropriate vector.
  • the type of vector used in the present invention is not particularly limited.
  • an autonomously replicating vector Eg, plasmid
  • it may be integrated into the genome of the host cell when introduced into the host cell and replicated along with the integrated chromosome.
  • the vector used in the present invention is an expression vector.
  • the gene of the present invention is operably linked to elements required for transcription (for example, a promoter and the like).
  • the promoter is a DNA sequence showing transcription activity in a host cell, and can be appropriately selected depending on the type of the host.
  • the promoters operable in bacterial cells include Bacillus stearothermophius us maltogenic amylase gene, Bacillus stearothermophius us maltogenic amylase gene, and Bacillus stearothermophius us maltogenic amylase gene.
  • promoters operable in mammalian cells include the SV40 promoter, the MT-1 (metamouth thionein gene) promoter, or the adenovirus 2 major late promoter.
  • promoters operable in insect cells include the polyhedrin promoter, the P10 promoter, the autographer's californica-polyhedrosis basic protein promoter, the baculourovirus immediate-early gene 1-port motor, and the baki Eurovirus 39K There is a delayed-type early gene promoter.
  • promoters operable in yeast host cells include promoters derived from yeast glycolysis genes, alcohol dehydrogenase gene promoters, TPI1 promoters, ADH2-4C promoters, and the like.
  • promoters operable in filamentous fungal cells include the ADH3 promoter or There is a tpi A promoter.
  • the DNA of the present invention may also be operably linked to a suitable terminator, such as, for example, a human growth hormone terminator or, for fungal hosts, a TPI1 terminator or an ADH3 terminator.
  • a suitable terminator such as, for example, a human growth hormone terminator or, for fungal hosts, a TPI1 terminator or an ADH3 terminator.
  • the recombinant vector of the present invention further comprises a polyadenylation signal (eg, from the SV40 or adenovirus 5E1b region), a transcription enhancer sequence (eg, the SV40 enhancer) and a translation enhancer sequence (eg, the adenovirus VA RNA). May be included.
  • the recombinant vector of the present invention may further comprise a DNA sequence that allows the vector to replicate in a host cell, such as the SV40 origin of replication (where the host cell is a mammalian cell). At the time of).
  • a host cell such as the SV40 origin of replication (where the host cell is a mammalian cell). At the time of).
  • the recombinant vector of the present invention may further contain a selection marker.
  • Selection markers include, for example, genes whose complement is lacking in the host cell, such as dihydrofolate reductase (DHFR) or the Schizosaccharomyces bombi TPI gene, or ampicillin, kanamycin, tetracycline, chlora Drug resistance genes such as mufenicol, neomycin or hygromycin can be mentioned.
  • DHFR dihydrofolate reductase
  • Schizosaccharomyces bombi TPI gene or ampicillin, kanamycin, tetracycline, chlora
  • Drug resistance genes such as mufenicol, neomycin or hygromycin can be mentioned.
  • Transformants can be prepared by introducing the DNA or recombinant vector of the present invention into a suitable host.
  • the host cell into which the DNA or recombinant vector of the present invention is introduced may be any cell as long as it can express the DNA construct of the present invention, and includes bacteria, yeast, fungi, and higher eukaryotic cells.
  • bacterial cells include Gram-positive bacteria such as Bacillus or Streptomyces or Gram-negative bacteria such as Escherichia coli. Transformation of these bacteria may be carried out by protoplast method or by using a competent cell by a known method.
  • Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells or CHO cells. Methods for transforming a mammalian cell and expressing the DNA sequence introduced into the cell are also known. For example, an electoral port method, a calcium phosphate method, a lipofection method and the like can be used.
  • yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, such as Saccharomyces cerevislae and Saccharomyces kluyveri.
  • Examples of a method for introducing a recombinant vector into a yeast host include an electoral poration method, a spheroblast method, and a lithium acetate method.
  • filamentous fungi such as cells belonging to Aspergillus, Neurospora, Fusarium, or Trichoderma.
  • transformation can be performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be performed according to known methods, for example, by homologous recombination or heterologous recombination.
  • the recombinant gene transfer vector and baculovirus are co-transfected into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is transmitted to the insect cells. Then, the protein can be expressed (for example, described in Baculovirus Expression Vectors, A Laboratory Manual; and current “Protocols” in “Molecular. Biology, Bio / Technology, 6, 47 (1988)).
  • Baculoviruses include, for example, Autographa, a virus that infects insects of the family Capitaridae * Californi, nuclei, polyhedrosis, virus (Autographs californica nuclear polyhedrosis virus) and the like.
  • Insect cells include the ovary cells of Spodoptera frugiperda, Sf9, Sf21 Recuroinoles 'Expression' Vectors, A 'Laboratory' Manual, Double H 'Freeman' and 'Company' (WH Freeman and Company) ), New York, (1992)], and Trichoplusia ni ovarian cells, Hi Five (Invitrogen), and the like.
  • Examples of a method for co-introducing a recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus include a calcium phosphate method and a lipofection method.
  • the above transformants are cultured in a suitable nutrient medium under conditions that allow expression of the introduced DNA construct.
  • a conventional protein isolation and purification method may be used.
  • the enzyme of the present invention when expressed in a dissolved state in the cells, after the culture is completed, the cells are collected by centrifugation, suspended in an aqueous buffer, and then disrupted by an ultrasonic disrupter or the like. Obtain a cell-free extract.
  • a normal protein isolation and purification method that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as getylaminoethyl (DEAE) Sepharose, cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia), butyl sepharose, phenol Hydrophobic chromatography using a resin such as Sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, etc. Used in combination, a purified sample can be obtained.
  • a resin such as getylaminoethyl (DEAE) Sepharose
  • cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia)
  • butyl sepharose phenol Hydrophobic
  • Example Example 1 O-glycan ⁇ 2, 8-sialyltransferase
  • 3'-sialyllactose, 6'-sialyl-jV-acetyllactosamine was purchased from Calbiochem.
  • -Acetylneuraminic acid (NeuAc), G4, Gal, N-Acetylgalactosamine (GalNAc) was purchased from Wako Pure Chemical Industries.
  • GD3 was purchased from Snow Brand Milk Products.
  • GQlb was purchased from Alexis Biochemicals.
  • CMP-[ 14 C]-NeuAc (12.0 GBq / mmol) was purchased from Amersham Pharmacia Biotech.
  • Sialidase (NANase II, III) was purchased from Glyko Inc.
  • V-glycanase (Glycopeptidase F) was purchased from Takara Shuzo.
  • [a- 32 P] dCTP was purchased from NEN. H Multiple tissue cDNA panel was purchased from Clontech. GMlb and its positional analog, GS-68,2,3-I sia ⁇ ylparagloboside (2,3-I SPG) and 2, D-sialylparagloboside (2,6-SPG) were obtained from Professor Kiso Makoto (Faculty of Agriculture, Gifu University) For NeuAc ⁇ 2, 3Gal and NeuAc ⁇ 2, 6Gal, those donated by Dr. Hideki Ishida (Noguchi Laboratory) were used. The anti-GD3 monoclonal antibody KM641 used was donated by Drs. Kyowa Hakko, Kenya Shitara and Chen Yu Hanai.
  • Anti-NeuAc a 2, 8 NeuAc a 2, 3Gal antibody S2-566 was purchased from Seikagaku Corporation.
  • Peroxidase-conjugated Aff iPure goat anti-mouse IgG + IgM H + L purchased from Jackson Research No Research, Inc.
  • BSM ⁇ ; 1-acid glycoprotein, ovomucoid deamination (Asia mouth) glycoprotein
  • This protein showed 42.0% and 38.3% homology with ST8Sia I and V among known mouse sialyltransferases at the amino acid sequence level, respectively (Fig. 2A). Since this protein had ⁇ 2,8-sialyltransferase activity as shown below, it was named i ⁇ glycan ⁇ 2,8_sialyltransferase ST8Sia VI of the present invention.
  • FIG. 2B shows the sequence information of human ST8Sia VI.
  • ST8Sia VI of mouse and human showed 82.4% homology at the amino acid sequence level (FIG. 2B).
  • P cDSA-mST8Sia VI and pcDSA- hST8Sia VI is Shigunanore peptides of each mouse immunoglobulin down IgM and Staphylococcus aureus protein A, and a mouse or human ST8Sia VI of the active domain (mouse ST8Sia VI in Amino acid numbers 26-398, Human ST8Sia VI encodes a secretory fusion protein consisting of amino acids 68-398).
  • the cells were transiently expressed in COS-7 cells (Kojima, N. et al. (1995) FEBS Lett. 360, 1-4).
  • the proteins of the present invention secreted extracellularly from the cells into which the respective expression vectors were introduced were named PA-mST8Sia VI (mouse) and PA-hST8Sia VI (human).
  • PA-mST8Sia VI and PA_hST8Sia VI were adsorbed on IgG-Sepharose (Amersham Pharmacia Biotech) and recovered from the medium. Sialyltransferase activity was performed as follows according to the method of Lee et al.
  • glycoproteins analysis was performed by SDS-polyacrylamide gel electrophoresis. The radioactivity was visualized and quantified using a BAS2000 radio image analyzer (Fujifilm). Table 1 shows the substrate specificity of PA-mST8Sia VI and PA-hST8Sia VI.
  • PA-mSrr8SiaVI and PA-hS8SiaVI were used to examine the specificity for various receptor substrates.
  • concentration of each substrate was 0.5 mg / ml for glycolipids and 1 mg / ml for glycoproteins, monosaccharides and oligosaccharides.
  • the relative activity was calculated based on the Fetuin uptake value PA-mST8Sia VI of 2.06 pmol / h / (ml enzyme solution PA-S8SiaVI of 0.204 pmol / h / (ml enzyme solution)).
  • R means the remaining sugar chain part of the W-type sugar chain. ND: Not measured.
  • NeuAca2,3Gai i, 3 (NeuAca2,6) GalNAc-0-Ser Thr
  • GlcNAcP 1,3 (NeuAca2,6) GalNAc-0-Ser hr
  • PA-mST8Sia VI is called Ne U A Ca 2,3 (6) Gal- at the non-reducing end, such as GM4, GM3, GDla, GTlb, GMlb, GSC-68, 2, 3-SPG, 2, 6-SPG It showed activity on glycolipids having a structure.
  • GM3 When GM3 is used as a substrate, the reaction product is cleaved by sialidase (NANase II), which specifically cleaves sialic acid bound by ⁇ 2,3-, ⁇ 2,6-linkage.
  • sialidase (NANase III) which specifically cleaves sialic acid bound by ⁇ 2,3-, a 2,6-, ct 2,8_, a 2,9-bond, was cut off (Fig. 3A).
  • Sialic acid was introduced into this reaction product via ⁇ 2,8 linkage by TLC immunostaining using the anti-GD3 monoclonal antibody KM641 (Saito, M. et al. (2000) Biochim. Biophys. Acta 1523, 230-235).
  • the confirmed GD3 was confirmed (Fig. 3B), indicating that PA-mST8Sia VI transfers sialic acid in an ⁇ 2,8 binding mode.
  • PA-mST8Sia VI when glycoprotein was used as a substrate (Table 1), PA-mST8Sia VI showed the highest activity against BSM containing only type-glycans. It also showed activity against type sugar chains, Fetuin containing type sugar chains, and Ovomucoid containing only type sugar chains, but the activity against Ovomucoid was lower than that of proteins containing type sugar chains. In addition, PA-mST8Sia VI did not show any activity against asia oral glycoprotein. In addition, experiments using monosaccharides and oligosaccharides as substrates (Table 1) revealed that the minimum sugar chain unit recognized by PA-mST8Sia VI as a substrate was NeuAco; 2, 3 (6) Gal. .
  • mouse ST8Sia VI the enzyme activity in the cells of the full-length clone was also examined (Fig. 5).
  • a 1.4 kb oil- ⁇ oal fragment containing the entire region encoding mouse ST8Sia VI was inserted into the o-site of the expression vector pRc / CMV and named pRc / CMV-ST8Sia VI. This was introduced into COS-7 cells using lipofectamine.
  • Gangliosides were extracted from these cells and subjected to TLC immunostaining using the monoclonal antibody S2-566 recognizing the NeuAc a 2,8 NeuAc ⁇ 2,3Gal structure (Fig.5A) .
  • the cells transfected with pRc / CMV-ST8Sia VI It was found that the amount of gangliosides having a NeuAc ⁇ 2,8NeuAc ⁇ 2,3Gal structure was significantly increased.
  • the intracellular glycoprotein in the cells transfected with pRc / CMV-ST8Sia VI, new NeuAco2,8NeuAca2,3Gal structures were formed on the type sugar chains (Fig. 5B). The above results indicate that mouse ST8Sia VI functions as ⁇ 2,8-sialyltransferase in vivo.
  • mouse ST8Sia VI is mainly expressed in kidney, heart, spleen, etc.
  • human ST8Sia VI is mainly expressed in various organs of the placenta and fetus, and various tumor cells.
  • Fig. 6B Example 2: j3-galactoside ⁇ 2,6-sialyltransferase
  • the reagents and samples used in the specific examples of the present invention are as follows. Fetuin, MN US asialofetuin, bovine submaxillary mucin (BSM), ol-acid glycoprotein, ovomucoid, lactosyl ceramide (LacCer), GA1, GM3, GMla, Gai i, 3GalNAc, Galpl, 3GlcNAc, Galpl, 4GlcNAc, Triton CF-54, ⁇ -lacto (Bovine testes) was purchased from Sigma. Paragloboside and rata tose were purchased from Wako Pure Chemical.
  • CMP- [ 14 C] -NeuAc (12.0 GBq / mmol) was purchased from Amersham Pharmacia Biotech. Lacto- ⁇ -tetraose, Lacto- ⁇ -neotetraose, and sialidase (NANase I, II) were purchased from Glyko Inc. [a- 32 P] dCTP was purchased from NEN. Human and mouse Multiple tissue cDNA panels were purchased from Clontech. BSA, al-acid glycoprotein, and ovomucoid desialylated glycoside glycoproteins were prepared by treating them in 0.02N HC1 at 80 ° C for 1 hour.
  • This cDNA had a single translation region encoding a type II membrane protein consisting of 529 amino acids and having a predicted molecular weight of 60,157.
  • the transmembrane domain was predicted to be present in the region of amino acids 12 to 30 according to the hydrophobic distribution diagram (FIG. 7B).
  • the amino acid sequence of this protein contains a sialyl motif conserved by sialyltransferase.
  • This protein showed the highest homology (48.9%) at the amino acid level with ST6Gal I among known human sialyltransferases (Fig. 9A), it did not differ from other families of sialyltransferases. It showed only -36% homology.
  • this protein had a / 3-galactosidyl 2,6-sialyltransferase activity.
  • the enzyme was named ST6Gal II.
  • human ST6Gal II there was also a short form clone with a different sequence in the middle of sialyl motif S, which is considered to be a splicing variant (Fig. 7A).
  • FIG. 8A shows the sequence information of mouse ST6Gal II.
  • Mouse ST6Gal II is 524 It was composed of amino acids, and the portion corresponding to the stem region was about 5 amino acids shorter than that of human ST6Gal II.
  • transmembrane domain of this protein was predicted to be present in the region of amino acid number 12-30 from the hydrophobic distribution diagram (FIG. 8B).
  • Human and mouse ST6Gal II showed 77.1% homology at the amino acid sequence level (FIG. 9B).
  • pcDSA-hST6GalII This was inserted into the 3 ⁇ 4oI site of the mammalian expression vector pcDSA.
  • This expression vector was named pcDSA-hST6GalII.
  • mouse ST6Gal II the synthetic DNA used for the clawing described above, 5′-CAATGAAACCACACTTGAAGCAATGGCGAC-3 ′ (corresponding to base numbers 1-30 in FIG. 8A) (SEQ ID NO: 23) Synthetic DNA containing Miwl site, 5'-CATCCAATTGACCAACAGCAATCCTGCGGC-3 '(corresponding to base numbers 83-112 in Fig. 8A) (SEQ ID NO: 26) using mouse ST6Gal II stem region and activity A MunHhol fragment encoding the domain was prepared. This was inserted into the coRI-JoI site of pcDSA and named the expression vector pcDSA-mST6GalII.
  • pcDSA-hST6Gal II and pcDSA-mST6Gal II are the immunoglobulin IgM signapeptide and Staphylococcus aureus protein A, and the active domain of mouse or human ST6Gal II (amino acids 33-529 in human ST6Gal II, respectively).
  • ST6Gal II a secretory fusion protein consisting of amino acids 31-524) is encoded.
  • the cells were transiently expressed in COS-7 cells (Kojima, N. et a J. (1995) FEES Lett. 360, 1-4).
  • the proteins of the present invention secreted extracellularly from the cells into which the respective expression vectors were introduced were named PA-hST6Gal II (human) and PA-mST6Gal II (mouse).
  • PA-hST6Gal II and PA-mST6Gal II were adsorbed on IgG-Sepharose (Amersham Pharmacia Biotech) and recovered from the medium.
  • Sialyltransferase activity was performed as follows according to the method of Lee et al. (Lee, Y.-C.
  • Table 2 shows the substrate specificity of PA_hST6GalII and PA-mST6GalII.
  • PA-hST6Gal II and PA-mST6Gal II were used to examine their specificity for various substrates.
  • concentration of each substrate was 0.5 mg / ml for glycolipids and 1 mg / ml for glycoproteins, monosaccharides and oligosaccharides.
  • the relative activity was calculated with the incorporation value of Galpl, 4GlcNAc as 100.
  • R means the remaining sugar chain part of the W-type sugar chain.
  • sialic acid introduced into the reaction product is sialic acid bound by ct2,3-linkage as in ST6GalI.
  • a sialidase that specifically cleaved sialic acid (NANase II) was not cleaved by sialidase that specifically cleaves acid (NAase I), but specifically cleaves sialic acid bound by ⁇ 2,3-, ⁇ 2,6-linkage. ) was cut (Fig. 11A).
  • this reaction product showed the same mobility as that of 6, -sialyl- -acetyllactosamine in TLC, and no change was observed in the mobility of TLC after galactosidase treatment (Fig. 11 ⁇ ). It was considered to be 6'-sialyl-acetylacetyltosamine in which sialic acid was introduced into galactose via ⁇ 2,6 bond. From the above, it was clarified that ST6Gal II transfers sialic acid to galactose in the ⁇ 2,6-linkage mode.
  • a particularly preferred substrate was considered to be an oligosaccharide having a Gal j31,4G1 C NAc structure at the non-reducing end.
  • ST6Gal I-specific primers (5′_TTATGATTCACACCAACCTGAAG-3 ′ (SEQ ID NO: 27) and 5′-CTTTGTACTTGTTCATGCTTAGG-3 ′ (SEQ ID NO: 28), The size of the PCR amplified fragment is 372 bp) and ST6Gal II specific primers (5, -AGACGTCATTTTGGTGGCCTGGG-3, (corresponding to base number 1264-1286 in Fig. 7A) (SEQ ID NO: 29)) and 5'-TTAAGAGTGTGGMTGACTGG- 3 ' (Corresponding to base numbers 1745 to 1765 in FIG.
  • the present invention O- glyca na 2 as a new enzyme, 8-sialyltransferase and novel protein that is secreted outside of the cell has an active portion of the enzyme is provided by. Since the enzyme and protein of the present invention have 0-glycana 2,8-sialyltransferase activity, they are useful, for example, as reagents for introducing human sugar chains into proteins. Also, O of the present invention - glycan ⁇ 2, 8- sialyltransferase is useful as a medicament for the treatment of genetic disorders that lack specific sugar chain human.
  • the glycan o; 2,8-sialyltransferase of the present invention can also be used as a medicament for the purpose of suppressing cancer metastasis, preventing virus infection, suppressing inflammatory response, and activating nerve tissue.
  • the ⁇ -glycan ⁇ 2,8-sialyltransferase of the present invention is useful as a research reagent or the like for increasing a physiological action by adding sialic acid to a drug or the like.
  • the present invention provides ⁇ -galactoside ⁇ 2,6-sialyltransferase as a novel enzyme and a novel protein having an active portion of the enzyme and secreted extracellularly.
  • the enzyme and the protein of the present invention comprise a 3 / 3-galactoside ⁇ 2,6-sialyltransferase Due to its activity, it became possible to introduce sialic acid more selectively into a galactose such as an oligosaccharide having a Gal / 31,4GlcNAc structure in an ⁇ 2,6 binding mode.
  • the / 3-galactoside ⁇ 2,6-sialyltransferase ST6Gal II of the present invention is a therapeutic drug for hereditary diseases lacking the specific sugar chain synthesized by this enzyme, and also suppresses cancer metastasis, virus infection, It is useful as a drug that has an inflammatory response inhibitory or neuronal activation effect, or as a research reagent that increases the physiological action by adding sialic acid to sugar chains, or inhibits the degradation activity of glycolytic enzymes. .

Abstract

It is intended to provide an O-glycan α 2,8-sialyltransferase having a novel substrate specificity and a substrate selectivity and a β-galactoside α 2,6-sialyltransferase having a novel substrate specificity and a substrate selectivity. These sialyltransferases are usable as drugs for inhibiting cancer metastasis, preventing viral infection, inhibiting inflammation and potentiating nerve tissue.

Description

明細書  Specification
糖鎖合成酵素 技術分野  Sugar chain synthase technical field
本発明は糖鎖合成酵素および該酵素をコードする D N Aに関するものである。 さらに詳しくは、 本発明はムチンなどの O型糖鎖のうち、 末端に Sia o; 2，3 (6) Gal (Sia : シアル酸、 Gal : ガラク トース）構造をもつ糖鎖のシアル酸部分にひ 2, 8 の 結合様式でシアル酸を効率良く転移する酵素（0- glycan α 2, 8-シアル酸転移酵 素、 ST8Sia VI)および該酵素をコードする D N A；並びに、 オリゴ糖などの糖鎖 のうち、 末端に Gal 1, 4GlcNAc (Gal : ガラク トース、 GlcNAc : -ァセチルグル コサミン） 構造をもつ糖鎖のガラク トース部分に α 2, 6 の結合様式でシアル酸を 効率良く転移する酵素 （ST6Gal II)および該酵素をコードする D N Aに関するも のである。 本発明の O- glycan ct 2, 8-シアル酸転移酵素および ]3 —ガラクトシド α 2 , 6—シアル酸転移酵素は、 癌転移抑制、 ウィルス感染抑防止、 炎症反応抑 制、 神経細胞賦活効果を有する薬剤として、 あるいは糖鎖にシアル酸を付加する ことにより生理作用を増加させるための試薬、 その他、 酵素阻害剤等として有用 である。 背景技術 The present invention relates to a sugar chain synthase and a DNA encoding the enzyme. More specifically, the present invention relates to the sialic acid moiety of a sugar chain having a Sia o; 2,3 (6) Gal (Sia: sialic acid, Gal: galactose) structure at the end of an O-type sugar chain such as mucin. An enzyme (0-glycan α2,8-sialyltransferase, ST8Sia VI) that efficiently transfers sialic acid in a binding mode of 2,8, and DNA encoding the enzyme; and sugar chains such as oligosaccharides of, Gal 1 to end, 4GlcNAc (Gal: galactose, GlcNAc: - Asechiruguru Kosamin) 2 alpha to galactose moiety of a sugar chain having a structure, 6 enzymes that efficiently transfers sialic acid binding modes (ST6Gal II ) And DNA encoding the enzyme. The O-glycan ct 2,8-sialyltransferase and] 3-galactoside α2,6-sialyltransferase of the present invention can suppress cancer metastasis, prevent virus infection, suppress inflammatory response, and activate nerve cells. It is useful as a drug having the same, or as a reagent for increasing a physiological action by adding sialic acid to a sugar chain, or as an enzyme inhibitor. Background art
シアル酸は、 たとえば細胞-細胞間伝達、 細胞基質相互作用、 細胞接着などの重 要な生理作用を司る物質である。 発生、 分化の過程に特異的な、 あるいは臓器特 異的なシアル酸含有糖鎖の存在が知られている。 シアル酸は糖タンパク質および 糖脂質の糖鎖部分の末端位置に存在しており、 これらの部位へのシアル酸の導入 は、 酵素的に CMP- Siaからの転移によってなされる。  Sialic acid is a substance that controls important physiological actions such as cell-cell communication, cell-substrate interaction, and cell adhesion. It is known that sialic acid-containing sugar chains specific to the process of development and differentiation or organ-specific are present. Sialic acid exists at the terminal position of the sugar chain portion of glycoproteins and glycolipids, and the introduction of sialic acid into these sites is performed by enzymatic transfer from CMP-Sia.
このシアル酸の酵素的導入 （シアル酸転移） を担う酵素は、 シアル酸転移酵素 (sialyltransferase)と呼ばれるグリコシルトランスフェラーゼ類である。ほ乳類 では現在までに 18種類のシアル酸転移酵素の存在が知られているが、これらはシ アル酸の転移様式から 4 つのフアミ リーに大別される（Tsuji, S. (1996) J. Biochem. 120, 1-13) 。 すなわち、 α 2, 3 の結合様式でガラク トースにシアル酸 を転移する α 2, 3-シアル酸転移酵素（ST3Gal-フアミリー）、 α 2, 6の結合様式でガ ラタ トースにシアル酸を転移する α 2， 6-シアル酸転移酵素（ST6Gal-フアミ リー）、 α 2, 6の結合様式で N-ァセチルガラタ トサミンにシアル酸を転移する GalNAc a 2, 6-シアル酸転移酵素（ST6GalNAc-ファミリー）、 および α 2, 8 の結合様式でシァ ル酸にシアル酸を転移する c 2, 8-シアル酸転移酵素（ST8Sia-フアミ リー）である。 このうち α 2， 8-シアル酸転移酵素については現在までに 5 種類の酵素（ST8Sia I - V)について cDNAクローユングが行われており、その酵素学的諸性質も明らかに なってレヽる（Yamamoto, A. et al. (1996) J. Neurochem. 66, 26-34 ; Kojima, N. et al. (1995) FEBS Lett. 360, 1—4 ; Yoshida, Y. et al. (1995) J. Biol. Chem. 270, 14628-14633； Yoshida, Y. et al. (1995) J. Biochem. 118, 658-664； Kono, M. et al. (1996) J. Biol. Chem. 271, 29366-29371)。 ST8Sia I はガンダリオ シドの GD3 合成酵素であり、 ST8Sia V は同じくガンダリオシドの GDlc, GTla, GQlb, GT3などを合成する酵素である。 ST8Sia II, IVは神経細胞接着分子（NCAM) の N型糖鎖上にポリシアル酸を合成する酵素である。 ST8Sia IIIは糖タンパク質 の N型糖鎖および糖脂質に見いだされる Sia a 2，3Gal ^ l，4GlcNAc 構造にシアル 酸を転移する酵素である。 これらの酵素はいずれも糖脂質あるいは N型糖鎖を好 ましい基質としており、 O型糖鎖に対する活性は、 NCAMの一つのアイソフォーム に見いだされる O型糖鎖上に ST8Sia II, IVがオリゴシアル酸/ポリシアル酸を合 成する例と、脂肪細胞特異的糖タンパク質 AdipoQの O型糖鎖に ST8SiaIIIが作用 する例が報告されているだけである （Suzuki, M. et al. (2000) Glycobiology 10, 1113；及ぴ Sato C， et al. (2001) J. Biol. Chem. 276, 28849-28856) 。 すなわ ち今までに報告されてきている α 2， 8-シアル酸転移酵素は、 通常 Ο型糖鎖を好ま しい基質としてはおらず、 これを好ましい基質とする a 2, 8-シアル酸転移酵素の 存在は知られていなかった。 The enzymes responsible for the enzymatic transfer of sialic acid (sialyltransferase) are glycosyltransferases called sialyltransferases. To date, there are 18 known sialyltransferases in mammals, It is roughly classified into four families according to the mode of transfer of lactic acid (Tsuji, S. (1996) J. Biochem. 120, 1-13). Α2,3-sialyltransferase (ST3Gal-family), which transfers sialic acid to galactose in the α2,3 binding mode, transfers sialic acid to galatatoose in the α2,6 bonding mode. α2,6-sialyltransferase (ST6Gal-family), GalNAc a2,6-sialyltransferase (ST6GalNAc-family), which transfers sialic acid to N-acetylgalatatosamine in the α2,6 binding mode, And c2,8-sialyltransferase (ST8Sia-family) that transfers sialic acid to sialic acid in the binding mode of α2,8. Of these, cDNA cloning of α2,8-sialyltransferase has been performed for five types of enzymes (ST8Sia I-V) to date, and their enzymological properties have been clarified (Yamamoto , A. et al. (1996) J. Neurochem. 66, 26-34; Kojima, N. et al. (1995) FEBS Lett. 360, 1-4; Yoshida, Y. et al. (1995) J. Biol. Chem. 270, 14628-14633; Yoshida, Y. et al. (1995) J. Biochem. 118, 658-664; Kono, M. et al. (1996) J. Biol. Chem. 271, 29366- 29371). ST8Sia I is a GD3 synthase of gandarioside, and ST8Sia V is an enzyme that synthesizes GDlc, GTla, GQlb, GT3, etc. of gandarioside. ST8Sia II and IV are enzymes that synthesize polysialic acid on the N-type sugar chain of nerve cell adhesion molecule (NCAM). ST8Sia III is an enzyme which transfers sialic acid to Sia a 2,3Gal ^ l, 4GlcNAc structure found in N-glycans and glycolipids glycoproteins. All of these enzymes use glycolipids or N-glycans as preferred substrates, and the activity on O-glycans is based on the oligosaccharides of ST8Sia II and IV on the O-glycans found in one isoform of NCAM. Only examples of acid / polysialic acid synthesis and ST8SiaIII acting on the O-glycan of adipocyte-specific glycoprotein AdipoQ have been reported (Suzuki, M. et al. (2000) Glycobiology 10 Sato C, et al. (2001) J. Biol. Chem. 276, 28849-28856). In other words, the α2,8-sialyltransferase that has been reported so far is not usually a type II sugar chain as a preferred substrate. The existence of was unknown.
また、 3 -ガラクトシド α 2, 6-シアル酸転移酵素については現在までに 1種類の 酵素 （ST6Gal I) についてのみ cDNAクローユングが行われており、 その酵素学的 諸性質も明らかになつている （Hamamoto, T. and Tsuji, S. (2001) ST6Gal-I in Handbook of Glycosyl transferases and Related Genes (Taniguchi, N. et al. Eds. ) pP295- 300)。 ST6Gal Iは糖タンパク質、 オリゴ糖またはガンダリオシドな どの末端糖鎖部分に Gal β 1, 4GlcNAc構造をもつものに対して活性を示すが、 Gal β 1, 4GlcNAc 構造のほかにラタトース （Gal /3 1, 4Glc) や場合によっては Gal /3 1, 3GlcNAc 構造でも基質にすることができる基質特異性の広い酵素である。 基質 特異性が広いということは、例えば ST6Gal Iを利用した機能性オリゴ糖などの合 成の際に、 原材料に不純物が混入していると、 それらも基質となって副産物が生 じてしまう可能性が考えられる。 従ってこの問題を解決するためには、 基質特異 性に関してより選択性の高い酵素が要求される。しかし現在までに -ガラク トシ ド α 2, 6-シアル酸転移酵素活性をもち、 基質特異性に関してより選択性の高い哺 乳動物由来の酵素は知られていなかった。 発明の開示 In addition, one type of 3-galactoside α2,6-sialyltransferase has been CDNA cloning is performed only for the enzyme (ST6Gal I), and its enzymatic properties are also clear (Hamamoto, T. and Tsuji, S. (2001) ST6Gal-I in Handbook of Glycosyl transferases and Related Genes (Taniguchi, N. et al. Eds.) P P 295-300). ST6Gal I has activity on Galβ1,4GlcNAc structures such as glycoproteins, oligosaccharides and gandariosides, but has the activity of Galβ1,4GlcNAc as well as ratatose (Gal / 31, It is an enzyme with a wide substrate specificity that can be used as a substrate even with 4Glc) or, in some cases, Gal / 31 / 3GlcNAc structure. The wide substrate specificity means that, for example, when synthesizing functional oligosaccharides using ST6Gal I, if impurities are mixed in the raw materials, they may also become substrates and generate by-products. Sex is considered. Therefore, to solve this problem, enzymes that are more selective with respect to substrate specificity are required. But to date - galactosyl de alpha 2, has a 6-sialyltransferase activity, an enzyme derived from a more highly selective mammals animals regard substrate specificity has not been known. Disclosure of the invention
上記した通り、 今までに知られている α 2， 8-シアル酸転移酵素は 5種類存在す るが、 これらはいずれも Ν型糖鎖をもつ糖タンパク質またはガンダリオシドなど の糖脂質を主な基質とし、 Ο型糖鎖をもつ糖タンパク質に対しては活性を全く示 さないか、 限定的な活性を示すだけであった。 本発明の第一の目的は、 Ο型糖鎖 に対し高い活性を示す新規な O-glycan α 2， 8-シアル酸転移酵素を提供すること である。 また、本発明は、 0- glycan ct 2, 8-シアル酸転移酵素をコードする cDNA をクローニングし、 該 O- glycan α 2, 8-シアル酸転移酵素をコードする DNA配列 および該酵素のアミノ酸配列を提供することを目的とする。 さらに本発明は、 上 記の O-glycan α 2, 8-シアル酸転移酵素の構造のうち、 活性に係わる部分を大量 に蛋白として発現させることを目的とする。  As described above, there are five types of α2,8-sialyltransferases known so far, all of which use glycolipids such as glycoproteins with Ν-type sugar chains or gandariosides as main substrates. It showed no activity or only a limited activity on glycoproteins having type I sugar chains. A first object of the present invention is to provide a novel O-glycan α2,8-sialyltransferase having high activity on type I sugar chains. Further, the present invention provides a method for cloning a cDNA encoding 0-glycan ct 2,8-sialyltransferase, a DNA sequence encoding the O-glycan α2,8-sialyltransferase, and an amino acid sequence of the enzyme. The purpose is to provide. Another object of the present invention is to express a portion of the structure of the above-mentioned O-glycan α2,8-sialyltransferase involved in activity as a protein in a large amount.
さらにまた上記した通り、哺乳動物で今までに知られている ]3 -ガラク トシド α 2, 6-シアル酸転移酵素は 1種類 （ST6Gal I) だけである。 これは糖タンパク質、 ォリゴ糖またはガングリオシドなどの末端糖鎖部分に Gal β 1, 4GlcNAc 構造をも つものに対して活性を示すが、 Gal j3 1, 4GlcNAc 構造のほかにラクトース （Gal 1, 4Glc)や場合によっては Gal 1, 3GlcNAc構造でも基質にすることができる基質 特異性の広い酵素である。 本発明の第二の目的は、 この基質特異性が広いという 問題点を解決し、ォリゴ糖上の Gal β 1, 4GlcNAc構造に対してより選択性の高い基 質特異性を示す新規 ]3 -ガラク トシド α 2, 6-シアル酸転移酵素および該酵素をコ ードする DNAを提供することである。 Furthermore, as mentioned above, there is only one type of [3-galactoside α2,6-sialyltransferase (ST6Gal I) known so far in mammals]. This is a glycoprotein, Although it has activity against Gal β1, 4GlcNAc structure at the terminal sugar chain such as oligosaccharide or ganglioside, in addition to Gal j3 1,4GlcNAc structure, lactose (Gal 1, 4Glc) and sometimes Gal It is an enzyme with wide substrate specificity that can be used as a substrate even with 1,3GlcNAc structures. A second object of the present invention is to solve the problem of wide substrate specificity, and to provide a novel substrate specificity that shows higher selectivity for the Galβ1,4GlcNAc structure on oligosaccharides. An object of the present invention is to provide a galactoside α2,6-sialyltransferase and a DNA encoding the enzyme.
本発明者は、 上記の課題を解決すべく鋭意努力し、 マウス脳及び心臓の各 cDNA ライブラリーをスクリーニングし、 またマウス腎臓由来 cDNA を錶型とした PCR を行うことにより、 O - glycan ct 2, 8-シアル酸転移酵素をコ一ドする cDNAをクロ 一二ングすることに成功した。さらに、本発明者は、ヒ トシアル酸転移酵素 ST6Gal I のアミノ酸配列を用いて、 これと相同性を示す新規シアル酸転移酵素をコード してレ、るクローンを expressed sequence tag (dbEST)のデ^ τタベースで検索し、 GenBank™ accession Nos. BE613250, BE612797, BF038052の各 ESTクローンを取 得した。 またそれらの塩基配列情報を利用して、 dbEST とヒ トゲノムの High throughput genomic sequenceのデータベースを検索し、 関連 ESTクローンとゲ ノム遺伝子の塩基配歹' IJ情報を取得した。 以上の塩基配列情報をもとにポリメラー ゼ連鎖反応法 （PCR)用のプライマーを作製し、 ヒ ト大腸由来 cDNA を铸型として PCRを行い、 得られた増幅断片と入手 ESTクローン由来の DNA断片を連結するこ とによって翻訳領域全長を含むクローンを取得した。 そして、 該クローンにより コードされるタンパク質が /3—ガラク トシド α 2， 6 —シアル酸転移酵素活性を 有していることを確認した。 本発明はこれらの知見に基づいて完成したものであ る。  The present inventors have made intensive efforts to solve the above problems, screened mouse brain and heart cDNA libraries, and performed O-glycan ct 2 We succeeded in cloning cDNA encoding 8-, 8-sialyltransferase. Furthermore, the present inventor has used the amino acid sequence of the human sialyltransferase ST6Gal I to encode a novel sialyltransferase showing homology thereto, and cloned the clone with the expressed sequence tag (dbEST). Search was performed using a τ database to obtain EST clones of GenBank ™ accession Nos. BE613250, BE612797, and BF038052. Using these base sequence information, we searched dbEST and the high throughput genomic sequence database of the human genome, and obtained the base sequences of related EST clones and genomic genes. Based on the above base sequence information, primers for polymerase chain reaction (PCR) were prepared, and PCR was performed using human colon-derived cDNA as type III. The amplified fragment obtained and the DNA fragment derived from the obtained EST clone By ligation, a clone containing the entire translation region was obtained. Then, it was confirmed that the protein encoded by the clone had / 3-galactoside α2,6-sialyltransferase activity. The present invention has been completed based on these findings.
即ち、 本発明によれば、 以下の基質特異性および基質選択性を有することを特 徴とする、 Ο - glycan α 2, 8-シアル酸転移酵素が提供される。  That is, according to the present invention, there is provided Ο-glycan α2, 8-sialyltransferase, which has the following substrate specificity and substrate selectivity.
基質特異性：末端に Sia a 2, 3 (6) Gal (ここで、 Siaはシアル酸を示し、 Galは ガラク トースを示す)構造をもつ糖を基質とする ； 基質選択性：糖脂質および N型糖鎖よりも優先的に O型糖鎖に対してシアル酸 を取り込ませる ： Substrate specificity: using a sugar having a terminal Sia a 2, 3 (6) Gal (where Sia represents sialic acid and Gal represents galactose) substrate; Substrate selectivity: Incorporates sialic acid into O-glycans preferentially over glycolipids and N-glycans:
好ましくは、本発明により、下記の何れかのァミノ酸配列を有する O- glycan a 2, 8 -シアル酸転移酵素が提供される。  Preferably, the present invention provides an O-glycan a 2,8-sialyltransferase having any one of the following amino acid sequences.
( 1 ) 配列表の配列番号 1または 3に記載のァミノ酸配列；又は  (1) an amino acid sequence according to SEQ ID NO: 1 or 3 in the sequence listing; or
( 2 ) 配列表の配列番号 1または 3に記載のァミノ酸配列において 1から数個の アミノ酸の欠失、置換及び 又は付加を有するアミノ酸配列を有し、 O - glycan a 2, 8-シアル酸転移を触媒する活性を有するアミノ酸配列：  (2) O-glycan a 2, 8-sialic acid having an amino acid sequence having deletion, substitution and / or addition of one to several amino acids in the amino acid sequence described in SEQ ID NO: 1 or 3 in the sequence listing. Amino acid sequence having activity to catalyze transfer:
本発明の別の側面によれば、 上記した本発明の O-glycan ひ 2， 8-シアル酸転移 酵素のアミノ酸配列をコードする O- glycan α 2, 8-シアル酸転移酵素遺伝子が提 供される。  According to another aspect of the present invention, there is provided an O-glycan α2,8-sialyltransferase gene encoding the amino acid sequence of the above-described O-glycan 2,8-sialyltransferase of the present invention. You.
好ましくは、本発明により、下記の何れかの塩基配列を有する O- glycan « 2, 8- シアル酸転移酵素が提供される。  Preferably, the present invention provides an O-glycan «2, 8- sialyltransferase having any one of the following nucleotide sequences.
( 1 ) 配列表の配列番号 2に記載の塩基配列中の塩基番号 7 7番目から 1 2 7 0 番目で特定される塩基配列；  (1) a nucleotide sequence specified by nucleotide numbers 77 to 127 in the nucleotide sequence of SEQ ID NO: 2 in the sequence listing;
( 2 ) 配列表の配列番号 2に記載の塩基配列中の塩基番号 7 7番目から 1 2 7 0 番目で特定される塩基配列において 1から数個の塩基の欠失、 置換及び Z又は付 加を有する塩基配列を有し、 0- glycan α 2, 8-シアル酸転移を触媒する活性を有 する蛋白質をコードする塩基配列：  (2) Deletion, substitution, Z or addition of one to several bases in the base sequence specified by base numbers 77 to 127 in the base sequence described in SEQ ID NO: 2 in the sequence listing A base sequence encoding a protein having an activity of catalyzing 0-glycan α2,8-sialyltransferase having the following sequence:
( 3 ) 配列表の配列番号 4に記載の塩基配列中の塩基番号 9 2番目から 1 2 8 5 番目で特定される塩基配列；  (3) a base sequence specified by base numbers 9 to 1285 in the base sequence described in SEQ ID NO: 4 in the sequence listing;
( 4 ) 配列表の配列番号 4に記載の塩基配列中の塩基番号 9 2番目から 1 2 8 5 番目で特定される塩基配列において 1から数個の塩基の欠失、 置換及び 又は付 加を有する塩基配列を有し、 0- glycan α 2, 8-シアル酸転移を触媒する活性を有 する蛋白質をコードする塩基配列：  (4) Deletion, substitution, and / or addition of one to several bases in the base sequence specified by base numbers 92 to 12285 in the base sequence described in SEQ ID NO: 4 in the sequence listing. A nucleotide sequence encoding a protein having an activity of catalyzing 0-glycan α2,8-sialyltransferase having the following nucleotide sequence:
本発明のさらに別の側面によれば、 上記した本発明の Ο - glycan α 2, 8 -シアル 酸転移酵素遣伝子を含む組み換えベクター （好ましくは、 発現ベクター） ；上記 した組み換えベクターにより形質転換された形質転換体；並びに上記した形質転 換体を培養し培養物から本発明の酵素を採取することを特徴とする本発明の酵素 の製造方法が提供される。 According to still another aspect of the present invention, a recombinant vector (preferably, an expression vector) containing the Ο-glycan α2,8-sialyltransferase gene of the present invention described above; And a method for producing the enzyme of the present invention, which comprises culturing the transformant and collecting the enzyme of the present invention from the culture.
本発明のさらに別の側面によれば、 下記の何れかのァミノ酸配列を有する o According to still another aspect of the present invention, it has any of the following amino acid sequences:
-glycan α 2, 8-シアル酸転移酵素活性ドメインから成る蛋白質が提供される。 a protein comprising -glycan α 2, 8-sialyltransferase active domain.
( 1 ) 配列表の配列番号 1に記載のァミノ酸配列のァミノ酸番号 2 6〜 3 9 8か ら成るアミノ酸配列；  (1) an amino acid sequence consisting of amino acid numbers 26-398 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing;
( 2 ) 配列表の配列番号 1に記載のァミノ酸配列のァミノ酸番号 2 6〜 3 9 8か ら成るアミノ酸配列において 1から数個のァミノ酸の欠失、 置換及び Ζ又は付加 を有するアミノ酸配列を有し、 O- glycan α 2, 8_シアル酸転移を触媒する活性を 有するアミノ酸配列：  (2) an amino acid having 1 to several amino acid deletions, substitutions and Ζ or additions in the amino acid sequence consisting of amino acid numbers 26 to 398 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing An amino acid sequence having an activity of catalyzing O-glycan α2,8_sialyltransferase:
( 3 ) 配列表の配列番号 3に記載のァミノ酸配列のァミノ酸番号 6 8〜 3 9 8カ ら成るアミノ酸配列；又は  (3) an amino acid sequence comprising amino acid numbers 68 to 398 of the amino acid sequence described in SEQ ID NO: 3 in the sequence listing; or
( 4 ) 配列表の配列番号 3に記載のァミノ酸配列のァミノ酸番号 6 8〜 3 9 8か ら成るアミノ酸配列において 1から数個のァミノ酸の欠失、 置換及び 又は付加 を有するアミノ酸配列を有し、 Ο- glycan a 2, 8_シアル酸転移を触媒する活性を 有するアミノ酸配列：  (4) an amino acid sequence comprising amino acid numbers 68 to 398 of the amino acid sequence described in SEQ ID NO: 3 in the sequence listing, the amino acid sequence having one to several amino acid deletions, substitutions, and / or additions An amino acid sequence having the activity of catalyzing the transfer of Ο-glycana 2,8_sialyl acid:
本発明のさらに別の側面によれば、 本発明の O- glycan α 2， 8 -シアル酸転移酵 素の活性ドメィンであるポリべプチド部分とシグナルぺプチドとを含む細胞外分 泌型の蛋白であって、 0- glycan α 2, 8-シアル酸転移を触媒する活性を有する蛋 白質が提供される。  According to still another aspect of the present invention, an extracellular secretory protein comprising a polypeptide portion, which is an active domain of the O-glycan α2,8-sialyltransferase of the present invention, and a signal peptide And a protein having an activity of catalyzing 0-glycan α2,8-sialyltransfer is provided.
本発明のさらに別の側面によれば、 上記した本発明の細胞外分泌型の蛋白質を コードする遺伝子が提供される。  According to still another aspect of the present invention, there is provided a gene encoding the above-mentioned extracellular secretory protein of the present invention.
本発明のさらに別の側面によれば、 上記した本発明の細胞外分泌型の蛋白質を コードする遺伝子を含む組み換えベクター （好ましくは、 発現ベクター） ；上記 した組み換えベクターにより形質転換された形質転換体；並びに上記した形質転 換体を培養し培養物から本発明の酵素を採取することを特徴とする本発明の蛋白 質の製造方法が提供される。 According to still another aspect of the present invention, a recombinant vector (preferably, an expression vector) containing a gene encoding the above-described extracellular secretory protein of the present invention; a transformant transformed with the above-described recombinant vector; And a protein of the present invention, which comprises culturing the above-mentioned transformant and collecting the enzyme of the present invention from the culture. A quality manufacturing method is provided.
本発明のさらに別の側面によれば、 以下の作用および基質特異性を有すること を特徴とする、 3—ガラク トシドひ 2， 6—シアル酸転移酵素が提供される。 According to still another aspect of the present invention, there is provided 3-galactoside 2,6-sialyltransferase, which has the following action and substrate specificity.
( 1 ) 作用 ； (1) action;
末端にガラク トース ] 3 1 , 4 N—ァセチルダルコサミン構造をもつ糖鎖のガラ ク トース部分に《 2, 6の結合様式でシアル酸を転移する。  Galactose at the end] 31 Sialic acid is transferred to the galactose portion of the sugar chain having a 1,4N-acetyl-dalcosamine structure in a << 2,6 binding mode.
(2) 基質特異性；  (2) substrate specificity;
末端にガラクトース ] 3 1, 4 N—ァセチルダルコサミン構造をもつ糖鎖を基質 とし、 ラク トース、 及び末端にガラク トース / 3 1 , 3N—ァセチルダルコサミン 構造をもつ糖鎖を基質としない。  [Galactose at the terminal] 31,4 N-Acetyldarcosamine structure is used as a substrate, and lactose and a sugar chain having a galactose / 31,3N-acetyldarcosamine structure at the end are not used as substrates. .
本発明のさらに別の側面によれば、 下記の何れかのァミノ酸配列を有する ]3 - ガラク トシド α 2, 6—シアル酸転移酵素が提供される。  According to still another aspect of the present invention, there is provided a 3-galactoside α2,6-sialyltransferase having any one of the following amino acid sequences:
( 1 ) 配列表の配列番号 5または 7に記載のァミノ酸配列；又は  (1) an amino acid sequence according to SEQ ID NO: 5 or 7 in the sequence listing; or
( 2 ) 配列表の配列番号 5または 7に記載のァミノ酸配列において 1から数個の アミノ酸の欠失、 置換及び Ζ又は付加を有するアミノ酸配列を有し、 ]3—ガラク トシド α 2， 6—シアル酸転移を触媒する活性を有するアミノ酸配列：  (2) has an amino acid sequence having deletion, substitution and Ζ or addition of one to several amino acids in the amino acid sequence described in SEQ ID NO: 5 or 7 in the sequence listing, and] 3-galactoside α2,6 —Amino acid sequence having activity to catalyze sialyl transfer:
本発明のさらに別の側面によれば、 上記した本発明の J3—ガラクトシドひ 2, 6—シアル酸転移酵素のアミノ酸配列をコードする 一ガラクトシド α 2， 6— シアル酸転移酵素遺伝子が提供される。  According to still another aspect of the present invention, there is provided a monogalactoside α2,6-sialyltransferase gene encoding the amino acid sequence of the above-mentioned J3-galactoside 2,6-sialyltransferase of the present invention. .
本発明のさらに別の態様によれば、 下記の何れかの塩基配列を有する i3—ガラ ク トシド α 2, 6—シアル酸転移酵素遣伝子が提供される。  According to still another aspect of the present invention, there is provided an i3-galactoside α2,6-sialyltransferase gene having any one of the following nucleotide sequences.
(1) 配列表の配列番号 6に記載の塩基配列中の塩基番号 1 76番目から 1 76 2番目で特定される塩基配列；  (1) a base sequence specified by base numbers 176 to 1762 in the base sequence described in SEQ ID NO: 6 in the sequence listing;
(2) 配列表の配列番号 6に記載の塩基配列中の塩基番号 1 7 6番目から 1 76 2番目で特定される塩基配列において 1から数個の塩基の欠失、 置換及び/又は 付加を有する塩基配列を有し、 ]3—ガラク トシド α 2， 6—シアル酸転移を触媒 する活性を有する蛋白質をコ一ドする塩基配列： ( 3 ) 配列表の配列番号 8に記載の塩基配列中の塩基番号 3番目から 1 5 7 4番 目で特定される塩基配列；又は (2) deletion, substitution and / or addition of one to several bases in the base sequence specified by base numbers 176 to 176 in the base sequence described in SEQ ID NO: 6 in the sequence listing A nucleotide sequence encoding a protein having an activity of catalyzing the transfer of 3-galactoside α2,6-sialyl acid: (3) a nucleotide sequence identified by nucleotide number 3 to nucleotide 157 in the nucleotide sequence of SEQ ID NO: 8 in the sequence listing; or
( 4 ) 配列表の配列番号 8に記載の塩基配列中の塩基番号 3番目から 1 5 7 4番 目で特定される塩基配列において 1から数個の塩基の欠失、 置換及び/又は付加 を有する塩基配列を有し、 /3—ガラク トシド α 2， 6—シアル酸転移を触媒する 活性を有する蛋白質をコードする塩基配列：  (4) Deletion, substitution and / or addition of one to several bases in the base sequence specified by the base number 3 to 157 4 in the base sequence described in SEQ ID NO: 8 in the sequence listing. A nucleotide sequence encoding a protein having an activity to catalyze the transfer of / 3-galactoside α2,6-sialyl acid having the following nucleotide sequence:
本発明のさらに別の側面によれば、 本発明の /3 _ガラク トシド α 2 , 6—シァ ル酸転移酵素遺伝子を含む組み換えべクターが提供される。  According to still another aspect of the present invention, there is provided a recombinant vector comprising the / 3_galactoside α2,6-sialyltransferase gene of the present invention.
本発明の組み換えベクターは、 好ましくは、 発現ベクターである。  The recombinant vector of the present invention is preferably an expression vector.
本発明のさらに別の側面によれば、 本発明の組み換えべクタ一により形質転換 された形質転換体が提供される。  According to still another aspect of the present invention, there is provided a transformant transformed by the recombinant vector of the present invention.
本発明のさらに別の側面によれば、 本発明の形質転換体を培養し培養物から本 発明の酵素を採取することを特徴とする、本発明の酵素の製造方法が提供される。 本発明のさらに別の側面によれば、 下記の何れかのァミノ酸配列を有する ]3 - ガラク トシド α 2 , 6—シアル酸転移酵素活性ドメインから成る蛋白質が提供さ れる。  According to still another aspect of the present invention, there is provided a method for producing the enzyme of the present invention, which comprises culturing the transformant of the present invention and collecting the enzyme of the present invention from the culture. According to still another aspect of the present invention, there is provided a protein comprising a 3-galactoside α2,6-sialyltransferase active domain having any one of the following amino acid sequences:
( 1 ) 配列表の配列番号 5に記載のァミノ酸配列のァミノ酸番号 3 3〜 5 2 9力 ら成るアミノ酸配列；  (1) an amino acid sequence consisting of amino acid numbers 33 to 529 of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing;
( 2 ) 配列表の配列番号 5に記載のァミノ酸配列のァミノ酸番号 3 3〜 5 2 9力 ら成るアミノ酸配列において 1から数個のァミノ酸の欠失、 置換及び Ζ又は付加 を有するアミノ酸配列を有し、 i3—ガラク トシド α 2 , 6—シアル酸転移を触媒 する活性を有するアミノ酸配列：  (2) an amino acid having 1 to several amino acid deletions, substitutions and Ζ or additions in the amino acid sequence consisting of amino acids 33 to 529 in the amino acid sequence described in SEQ ID NO: 5 in the sequence listing An amino acid sequence having the sequence and having an activity of catalyzing i3-galactoside α 2,6-sialyltransferase:
( 3 ) 配列表の配列番号 7に記載のァミノ酸配列のァミノ酸番号 3 1〜 5 2 4か ら成るアミノ酸配列；又は  (3) an amino acid sequence consisting of amino acid numbers 31 to 524 of the amino acid sequence described in SEQ ID NO: 7 in the sequence listing; or
( 4 ) 配列表の配列番号 7に記載のァミノ酸配列のァミノ酸番号 3 1〜 5 2 4カ ら成るアミノ酸配列において 1から数個のァミノ酸の欠失、 置換及びノ又は付加 を有するアミノ酸配列を有し、 /3—ガラク トシド α 2 , 6—シアル酸転移を触媒 する活性を有するアミノ酸配列： (4) an amino acid having an amino acid sequence consisting of amino acids Nos. 31 to 524 of the amino acid sequence described in SEQ ID NO: 7 in the sequence listing, which has one to several amino acid deletions, substitutions and no or additions Catalyzes the transfer of / 3-galactoside α2,6-sialyl acid Amino acid sequence having the following activities:
本発明のさらに別の側面によれば、 本発明の ]3—ガラク トシド α 2 , 6—シァ ル酸転移酵素の活性ドメインであるポリぺプチド部分とシグナルぺプチドとを含 む細胞外分泌型の蛋白であって、 /3—ガラク トシド α 2 , 6—シアル酸転移を触 媒する活性を有する蛋白質が提供される。  According to still another aspect of the present invention, an extracellular secretory type comprising a polypeptide portion, which is an active domain of the 3-galactoside α2,6-sialyltransferase of the present invention, and a signal peptide. Provided is a protein, which has an activity of catalyzing a / 3-galactoside α2,6-sialyltransferase.
本発明のさらに別の態様によれば、 上記した本発明の蛋白質をコードする遺伝 子が提供される。  According to still another aspect of the present invention, there is provided a gene encoding the above-described protein of the present invention.
本発明のさらに別の態様によれば、 上記した本発明の遺伝子を含む組み換えべ クタ一が提供される。  According to still another aspect of the present invention, there is provided a recombinant vector comprising the above-described gene of the present invention.
本発明の組み換えベクターは、 好ましくは、 発現ベクターである。  The recombinant vector of the present invention is preferably an expression vector.
本発明のさらに別の態様によれば、 本発明の組み換えベクターにより形質転換 された形質転換体が提供される。  According to still another aspect of the present invention, there is provided a transformant transformed with the recombinant vector of the present invention.
本発明のさらに別の態様によれば、 本発明の形質転換体を培養し培養物から本 発明の蛋白質を採取することを特徴とする、 本発明の蛋白質の製造方法が提供さ れる。 図面の簡単な説明  According to still another aspect of the present invention, there is provided a method for producing the protein of the present invention, which comprises culturing the transformant of the present invention and collecting the protein of the present invention from the culture. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 マウスおょぴヒ トの ST8Sia VI cDNAの塩基配列と予測ァミノ酸配列を 示す。 膜貫通ドメインは下線、 シァリルモチーフ Lは二重線、 シァリルモチーフ Sは破線で示してある。 シァリルモチーフ VSで保存されているヒスチジンとダル タミン酸は四角で囲ってある。 型糖鎖が結合すると予想されるァスパラギンに は上線を付してある。 A, マウス ST8Sia VI。 B, ヒ ト ST8Sia VI。  FIG. 1 shows the nucleotide sequence and the predicted amino acid sequence of ST8Sia VI cDNA from mouse mouse. The transmembrane domain is underlined, the sialyl motif L is shown as a double line, and the sialyl motif S is shown as a dashed line. Histidine and daltamic acid conserved in the sialyl motif VS are boxed. Asparagine to which type-glycans are expected to bind is underlined. A, mouse ST8Sia VI. B, human ST8Sia VI.
図 2は、 ァミノ酸配列の比較を示す。  FIG. 2 shows a comparison of the amino acid sequences.
Aは、 マウスシアル酸転移酵素 ST8Sia I, ST8Sia V， ST8Sia VIのアミノ酸配 列の比較を示す。 各シァル酸転移酵素間で保存されているアミノ酸は四角で囲つ てある。 シァリルモチーフ Lは二重線で、 シァリルモチーフ Sは破線で示してあ る。シァリルモチーフ VSで保存されているヒスチジンとグルタミン酸にはァスタ リスクを付してある。 A shows a comparison of the amino acid sequences of mouse sialyltransferases ST8Sia I, ST8Sia V, and ST8Sia VI. Amino acids conserved between each sialyltransferase are boxed. The Cyaryl motif L is shown by a double line, and the Cyaryl motif S is shown by a broken line. You. Histidine and glutamic acid conserved in the sialyl motif VS are marked with an asterisk.
Bは、 マウス（m)およびヒ ト（h)の ST8Sia VIのアミノ酸配列の比較を示す。 両 酵素間で保存されているアミノ酸は四角で囲ってある。  B shows a comparison of the amino acid sequences of ST8Sia VI in mouse (m) and human (h). Amino acids conserved between both enzymes are boxed.
図 3は、 結合特異性の解析を示す。  FIG. 3 shows an analysis of the binding specificity.
Aは、マウス ST8SiaVIの分泌型組み換えタンパク質 PA- raST8Sia VIにより GM3 を [14C]-NeuAc でシアル化し、 それを α2, 3_, α 2， 6-結合特異的なシァリダーゼ (NANase 11)、 a 2, 3- α2,6-, α 2, 8-, α 2， 9-結合特異的シァリダーゼ（NANase III)で処理した反応産物を HPTLCで展開（展開溶媒はク口口ホルム：メタノール： 0.02% CaCl2=55:45:10) した結果 （上段） 、 およぴヒ ト ST8Sia VI の分泌型組み 換えタンパク質 PA-hST8Sia VIにより 3，-sialyllactoseを [14C]- NeuAcでシアル 化し、 それを NANase II、 NANase IIIで処理した反応産物を HPTLCで展開 （展開 溶媒は 1-プロパノール： アンモニア水：水 =6: 1:2.5) した結果 （下段） を示す。 A, GM3 was sialylated with [ 14 C] -NeuAc by mouse ST8SiaVI secreted recombinant protein PA-raST8Sia VI, and it was converted to α2, 3_, α2, 6-specific sialidase (NANase 11), a2 , 3-α2,6-, α2,8, α2,9-linkage-specific reaction sialidase (NANase III) -treated reaction product is developed on HPTLC (developing solvent is porcine form: methanol: 0.02% CaCl 2 2 = 55: 45: 10) (upper panel), and 3, -sialyllactose was sialylated with [ 14 C] -NeuAc by secreted recombinant protein PA-hST8Sia VI of human ST8Sia VI, which was then nanased. The results obtained by developing the reaction products treated with II and NANase III by HPTLC (developing solvent: 1-propanol: aqueous ammonia: water = 6: 1: 2.5) are shown (lower).
Bは、 GM3を PA- mST8Sia VIによりシアル化した反応産物の TLC免疫染色の結 果を示す。 レーン 1, GD3 (1 .ug)； レーン 2, GM3 (1 μ g)； レーン 3， 反応産物。 抗 GD3モノクロ一ナノレ抗体 KMり 41およひ Peroxidase— conjugated anti-mouse丄 g(j + IgM (H+L)で反応させた後、 ECLで発色した。  B shows the result of TLC immunostaining of the reaction product of GM3 sialylated with PA-mST8Sia VI. Lane 1, GD3 (1 .ug); Lane 2, GM3 (1 μg); Lane 3, reaction product. After reaction with anti-GD3 monoclonal antibody KM-41 and peroxidase-conjugated anti-mouse 丄 g (j + IgM (H + L), color was developed with ECL.
図 4は、 ST8Sia III または ST8Sia VI によって [14C]- NeuAc を取り込ませた Fetuinを - glycanaseで処理した結果を示す。 [14C] -NeuAcを取り込ませた Fetuin を - glycanaseで処理し、 SDS- PAGEで解析後、 BAS2000ラジオイメージアナライ ザ一で可視化した。 FIG. 4 shows the result of treating Fetuin having [ 14 C]-NeuAc incorporated by ST8Sia III or ST8Sia VI with -glycanase. Fetuin incorporating [ 14 C] -NeuAc was treated with -glycanase, analyzed by SDS-PAGE, and visualized with a BAS2000 radio image analyzer.
図 5は、 C0S-7細胞においてマウス ST8Sia VI全長 cDNAを過剰発現させたとき の影響を示す。  FIG. 5 shows the effect of overexpressing mouse ST8Sia VI full-length cDNA in C0S-7 cells.
Aは、 抗 euAca2, 8NeuAca2, 3Gal抗体 S2-566を用いて Tし C免疫染色を行った 結果を示す。 レーン 1, GD3標準物質（0.5 g)； レーン 2, GQlb標準物質（0.5 μ g) ; レーン 3, コントロールの COS- 7細胞（30mg)から抽出した酸性糖脂質画分； レ ーン 4， マウス全長 ST8Sia VI発現ベクター pRc/CMV-ST8Sia VIを導入した C0S-7 細胞（30 mg)から抽出した酸性糖脂質画分。 A shows the results of T and C immunostaining performed using anti-euAca2, 8NeuAca2, and 3Gal antibody S2-566. Lane 1, GD3 standard (0.5 g); Lane 2, GQlb standard (0.5 μg); Lane 3, acidic glycolipid fraction extracted from control COS-7 cells (30 mg); Lane 4, mouse C0S-7 transfected with full-length ST8Sia VI expression vector pRc / CMV-ST8Sia VI Acid glycolipid fraction extracted from cells (30 mg).
Bは、 C0S-7細胞または PRc/CMV- ST8Sia VIを導入した COS- 7細胞からミクロ ソーム画分を調製し、 SDS- PAGE に供した後（45 g/レーン）、 PVDF膜に転写して S2- 566抗体を用いてウェスタンブロットを行った結果を示す。 レーン 1, コント ロールの C0S-7細胞から調製したミク口ソーム画分； レーン 2, pRc/CMV-ST8Sia VIを導入した C0S-7細胞から調製したミクロソーム画分； レーン 3, コントロー ルの COS- 7細胞から調製したミク口ソーム画分を -ダリカナーゼ処理したもの； レーン 4， pRc/CMV-ST8Sia VIを導入した COS- 7細胞から調製したミクロソーム画 分を -グリカナ一ゼ処理したもの。 ST8Sia VI cDNAの導入により生じた S2- 566 抗体に認識されるバンドの主なものについては、 アスタリスクを付してある。 図 6は、 マウスおよびヒ トの ST8Sia VI遺伝子の発現様式を示す。 B is a microsomal fraction prepared from COS- 7 cells transfected with C0S-7 cells or P Rc / CMV- ST8Sia VI, was subjected to SDS- PAGE (45 g / lane), transferred to PVDF membrane 13 shows the results of Western blotting using the S2-566 antibody. Lane 1, microsomal fraction prepared from control C0S-7 cells; lane 2, microsomal fraction prepared from C0S-7 cells transfected with pRc / CMV-ST8Sia VI; lane 3, control COS- Lane 4: Micosomal fraction prepared from 7 cells treated with daricanase; Lane 4, microsomal fraction prepared from COS-7 cells transfected with pRc / CMV-ST8Sia VI treated with -glycanase. The major bands recognized by the S2-566 antibody generated by the introduction of ST8Sia VI cDNA are marked with an asterisk. FIG. 6 shows the expression modes of ST8Sia VI gene in mouse and human.
Aは、 マウス各種臓器より調製した poly (A) + RNA (約 2 ^ g/レーン）を用いて マウス ST8Sia VI遺伝子の発現様式をノーザン解析した結果を示す。  A shows the results of Northern analysis of the expression mode of the mouse ST8Sia VI gene using poly (A) + RNA (about 2 ^ g / lane) prepared from various mouse organs.
Bは、 Multiple Tissue cDNA Panel (Clontech)を用いて PCR法によりヒ ト ST8Sia VI遺伝子の発現様式を解析した結果を示す。 ヒ ト ST8Sia VI特異的プライマーと して、 5' - CCAGTGTCCCAGCCTTTTGT-3' (図 1Bの塩基番号 608- 627に相当） （配列番 号 1 7 ) および 5' -TGAGTGGGGAAGCTTTGGTC-3' (図 1Bの塩基番号 1407-1426の相 補鎖に相当） （配列番号 1 8 ) を用いた（PCR増幅断片の大きさは 819 bp)。  B shows the result of analyzing the expression mode of the human ST8Sia VI gene by PCR using the Multiple Tissue cDNA Panel (Clontech). As human ST8Sia VI-specific primers, 5′-CCAGTGTCCCAGCCTTTTGT-3 ′ (corresponding to base numbers 608-627 in FIG. 1B) (SEQ ID NO: 17) and 5′-TGAGTGGGGAAGCTTTGGTC-3 ′ (base in FIG. 1B) No. 1407-1426) (SEQ ID NO: 18) was used (the size of the PCR amplified fragment was 819 bp).
図 7は、 ヒ ト ST6Gal II cDNAの塩基配列と予測ァミノ酸配列、 およびその疎水 性分布図を示す。  FIG. 7 shows the nucleotide sequence of human ST6Gal II cDNA, the predicted amino acid sequence, and its hydrophobicity distribution map.
Aは、 ヒ ト ST6Gal II cDNAの塩基配列と予測ァミノ酸配列を示す。 膜貫通ドメ インは下線、 シァリルモチーフ Lは二重線、 シァリルモチーフ Sは破線で示して ある。シァリルモチーフ VSで保存されているヒスチジンとグルタミン酸は四角で 囲ってある。 型糖鎖が結合すると予想されるァスパラギンには上線を付してあ る。  A shows the nucleotide sequence of human ST6Gal II cDNA and the predicted amino acid sequence. The transmembrane domain is underlined, the sialyl motif L is shown as a double line, and the sialyl motif S is shown as a dashed line. Histidine and glutamic acid conserved in the sialyl motif VS are boxed. Asparagine to which type-glycans are expected to bind is underlined.
Bは、 ヒ ト ST6Gal IIの疎水性分布図を示す。 N末端側の大きな疎水性領域は 膜貫通ドメインと予測される。 図 8は、 マウス ST6Gal II cDNAの塩基配列と予測ァミノ酸配列、 およびその疎 水性分布図を示す。 B shows a hydrophobic distribution map of human ST6Gal II. The large N-terminal hydrophobic region is predicted to be a transmembrane domain. FIG. 8 shows the nucleotide sequence and predicted amino acid sequence of mouse ST6Gal II cDNA, and the hydrophobic distribution map thereof.
Aは、 マウス ST6Gal II cDNAの塩基配列と予測ァミノ酸配列を示す。 膜貫通ド メインは下線、 シァリルモチーフ Lは二重線、 シァリルモチーフ Sは破線で示し てある。シァリルモチーフ VSで保存されているヒスチジンとグルタミン酸は四角 で囲ってある。 型糖鎖が結合すると予想されるァスパラギンには上線を付して ある。  A shows the nucleotide sequence of mouse ST6Gal II cDNA and the predicted amino acid sequence. Transmembrane domains are underlined, sialyl motifs L are double lines, and sialyl motifs S are dashed. Histidine and glutamic acid conserved in the sialyl motif VS are boxed. Asparagine to which type-glycans are expected to bind is underlined.
Bは、 マウス ST6Gal IIの疎水性分布図を示す。 N末端側の大きな疎水性領域 は膜貫通ドメインと予測される。  B shows a hydrophobic distribution map of mouse ST6Gal II. The large N-terminal hydrophobic region is predicted to be a transmembrane domain.
図 9は、 ァミノ酸配列の比較を示す。  FIG. 9 shows a comparison of the amino acid sequences.
Aは、 ヒ トシアル酸転移酵素 ST6Gal Iと ST6Gal IIのァミノ酸配列の比較を示 す。 両シアル酸転移酵素間で保存されているアミノ酸は四角で囲ってある。 シァ リルモチーフ Lは二重線で、 シァリルモチーフ Sは破線で示してある。 シァリル モチーフ VS で保存されているヒスチジンとグルタミン酸にはアスタリスクを付 してある。  A shows a comparison of the amino acid sequences of human ST6Gal I and ST6Gal II. Amino acids conserved between both sialyltransferases are boxed. The sialyl motif L is shown as a double line, and the sialyl motif S is shown as a dashed line. Histidine and glutamic acid conserved in the sialyl motif VS are marked with an asterisk.
Bは、 ヒ ト（h)およびマウス（m)の ST6Gal IIのアミノ酸配列の比較を示す。 両 酵素間で保存されているアミノ酸は四角で囲ってある。  B shows a comparison of the amino acid sequences of human (h) and mouse (m) ST6Gal II. Amino acids conserved between both enzymes are boxed.
図 1 0は、 オリゴ糖に対する活性を示す。 様々なオリゴ糖を基質（10 ^ag/レー ン）として酵素反応を行い、 その反応産物を HPTLCで解析 （展開溶媒は 1-プロパ ノール： アンモニア水：水 =6: 1:2.5) した結果を示す。  FIG. 10 shows the activity on oligosaccharides. Enzymatic reaction was performed using various oligosaccharides as substrates (10 ^ ag / lane), and the reaction products were analyzed by HPTLC (developing solvent: 1-propanol: ammonia water: water = 6: 1: 2.5). Show.
図 1 1は、 結合特異性の解析を示す。  FIG. 11 shows an analysis of binding specificity.
Aは、 ヒ ト ST6Gal I (上段） 、 ヒ ト ST6Gal II (中段） 、 およびマウス ST6Gal II (下段） を用いて Gal 3 l,4GlcNAcを [14C]-NeuAc でシアル化し （レーン 1) 、 それを α2, 3-結合特異的シァリダーゼ（NANase I, レーン 2)、 a 2, 3- α2,6-結 合特異的シァリダーゼ（NANase II, レーン 3)で処理した反応産物を HPTLCで展開 (展開溶媒は 1_プロパノール： アンモニア水：水 =6:1:2.5) した結果を示す。 Bは、 ヒ ト ST6Gal I (上段） 、 ヒ ト ST6Gal II (中段） 、 およびマウス ST6Gal II (下段） を用いて Gal l, 4GlcNAcを [14C] - NeuAc でシアル化し （レーン 1) 、 それを ]3 -ガラクトシダーゼで処理した反応産物 （レーン 2) 、 およびコントロー ルとして Gal j3 l, 4GlcNAcを β -ガラク トシダーゼで処理した後に酵素反応を行つ た試料（レーン 3) を HPTLCで展開（展開溶媒は 1-プロパノール：アンモニア水： 水 =6 : 1 : 2. 5) した結果を示す。 レーン 2のバンドがブロードなのは、 /3 -ガラク ト シダーゼ溶液中に含まれている高濃度の硫酸ァンモニゥムの影響による。 A, using human ST6Gal I (upper), human ST6Gal II (middle), and mouse ST6Gal II (lower), sialized Gal3l, 4GlcNAc with [ 14 C] -NeuAc (lane 1), and Was treated with α2,3-linkage specific sidase (NANase I, lane 2) and a2,3-α2,6-linkage specific sialidase (NANase II, lane 3). Indicates the result of 1_propanol: ammonia water: water = 6: 1: 2.5). B: human ST6Gal I (upper), human ST6Gal II (middle), and mouse ST6Gal Using II (lower), Gal l, 4GlcNAc was sialylated with [ 14 C] -Neuac (lane 1), treated with] -galactosidase (lane 2), and Gal j3 l, Samples obtained by enzymatic reaction after treating 4GlcNAc with β-galactosidase (lane 3) are developed by HPTLC (developing solvent is 1-propanol: aqueous ammonia: water = 6: 1: 2.5). . The broad band in lane 2 is due to the effect of the high concentration of ammonium sulfate contained in the / 3-galactosidase solution.
図 1 2は、 ヒ ト ST6Gal I, ST6Gal IIおよびマウス ST6Gal II遺伝子の発現パ ターンの解析を示す。 ヒ ト ST6Gal I, ST6Gal II特異的プライマーとヒ ト組織 (Α) またはヒ ト腫瘍細胞（Β)の Multiple tissue cDNA panel (Clontech)を用い、 両遺 伝子の発現パターンを PCR法で解析した。 PCRは 94度 1分、 50度 1分、 72度 1 分 30秒を 1サイクルとし、 Glyceraldehyde 3 - phosphate dehydrogenase (G3PDH) 遺伝子については 25サイクル、 ヒ ト ST6Gal I, ST6Gal II遺伝子については 40 サイクル行って、 反応産物をァガロースゲル電気泳動で解析した。 Sk. muscle, skeletal muscle ; P. bl. leukocyte, peripheral blood leukocyte。 Cは、 マウ ス ST6Gal IIの発現パターンを、 マウス ST6Gal II特異的プライマーとマウス組 織の Multiple tissue cDNA panel (Clontech)を用い、 PCR法で解析した結果を示 す。 発明を実施するための最良の形態  FIG. 12 shows the analysis of the expression patterns of the human ST6Gal I, ST6Gal II and mouse ST6Gal II genes. Expression patterns of both genes were analyzed by PCR using human ST6Gal I and ST6Gal II specific primers and a multiple tissue cDNA panel (Clontech) of human tissue (Α) or human tumor cells (Β). PCR was performed at 94 ° C for 1 minute, 50 ° C for 1 minute, 72 ° C for 1 minute and 30 seconds, and 25 cycles for Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene and 40 cycles for human ST6Gal I and ST6Gal II genes The reaction products were analyzed by agarose gel electrophoresis. Sk. Muscle, skeletal muscle; P. bl. Leukocyte, peripheral blood leukocyte. Panel C shows the results of PCR analysis of the expression pattern of mouse ST6Gal II using mouse ST6Gal II-specific primers and a mouse tissue Multiple tissue cDNA panel (Clontech). BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明の実施態様及び実施方法について詳細に説明する。  Hereinafter, an embodiment and an implementation method of the present invention will be described in detail.
( 1 ) 本発明の酵素及び蛋白質  (1) Enzymes and proteins of the present invention
本発明の O- glycan α 2, 8-シアル酸転移酵素は、 以下の基質特異性および基質 選択性を有することを特徴とする。  The O-glycan α2, 8-sialyltransferase of the present invention is characterized by having the following substrate specificity and substrate selectivity.
基質特異性：末端に Sia a 2, 3 (6) Gal (ここで、 Siaはシアル酸を示し、 Galは ガラクトースを示す)構造をもつ糖を基質とする ；  Substrate specificity: a sugar having a structure of Sia a 2, 3 (6) Gal (where Sia represents sialic acid and Gal represents galactose) at the terminal is used as a substrate;
基質選択性：糖脂質および N型糖鎖よりも優先的に O型糖鎖に対してシアル酸 を取り込ませる ： 上記した基質特異性及び基質選択性は、 本明細書に記載した実施例で取得され たマウスおよびヒ ト由来の O-glycan α 2， 8-シアル酸転移酵素について実証され た性質である。 本発明の O-glycan o; 2, 8-シアル酸転移酵素の由来はマウスおよ びヒ ト由来のものに限定されるものではなく、 同型の Ο-glycan α 2, 8-シァノレ酸 転移酵素が他の哺乳類の組織に存在し、 かつ、 それらの O-glycan α 2, 8-シアル 酸転移酵素が互いに高度の相同性を有していることは当業者に容易に理解され る。 Substrate selectivity: Incorporates sialic acid into O-glycans preferentially over glycolipids and N-glycans: The above-described substrate specificity and substrate selectivity are properties demonstrated for the mouse and human O-glycan α2,8-sialyltransferase obtained in the examples described in the present specification. The origin of the O-glycan o; 2,8-sialyltransferase of the present invention is not limited to those derived from mice and humans, and the same type of Ο-glycan α2, 8-sialoletransferase It is easily understood by those skilled in the art that is present in other mammalian tissues, and that the O-glycan α2,8-sialyltransferase has a high degree of homology to each other.
このような O-glycan α 2, 8-シアル酸転移酵素は、 上記した基質特異性及び基 質選択性を有することを特徴とするものであり、 すべて本発明の範囲に属するも のである。  Such O-glycan α2,8-sialyltransferase is characterized by having the above-described substrate specificity and substrate selectivity, and all belong to the scope of the present invention.
このような酵素としては、 哺乳類組織由来の天然型酵素やその変異体、 または 以下の実施例で作製したような Ο - glycan α 2， 8-シアル酸転移を触媒し、 遺伝子 組み換え技術により製造された細胞外分泌型蛋白質などを挙げることができる が、 これらはいずれも本発明の範囲に包含されるものである。  Examples of such enzymes include natural enzymes derived from mammalian tissues and mutants thereof, and に よ り -glycan α2,8-sialyltransfer such as those prepared in the examples below, and are produced by gene recombination techniques. And extracellular secretory proteins, which are all included in the scope of the present invention.
本発明の 0- glycan α 2， 8-シアル酸転移酵素の一例としては、 下記の何れかの アミノ酸配列を有する Ο - glycan α 2, 8 -シアル酸転移酵素が挙げられる。  An example of the 0-glycan α2,8-sialyltransferase of the present invention includes Ο-glycan α2,8-sialyltransferase having any of the following amino acid sequences.
( 1 ) 配列表の配列番号 1または 3に記載のァミノ酸配列；又は  (1) an amino acid sequence according to SEQ ID NO: 1 or 3 in the sequence listing; or
( 2 ) 配列表の配列番号 1または 3に記載のァミノ酸配列において 1から数個の アミノ酸の欠失、置換及び/又は付加を有するアミノ酸配列を有し、 0- glycan a 2, 8-シアル酸転移を触媒する活性を有するアミノ酸配列：  (2) having an amino acid sequence having deletion, substitution and / or addition of one to several amino acids in the amino acid sequence described in SEQ ID NO: 1 or 3 of the sequence listing, and having 0-glycan a 2, 8-sialic acid; Amino acid sequence having activity to catalyze acid transfer:
さらに、 本発明の O- glycan α 2, 8 -シアル酸転移酵素の活性ドメイン、 あるい はそのアミノ酸配列の一部を改変又は修飾して得られる O-glycan ct 2, 8-シアル 酸転移酵素活性を有する蛋白質は全て本発明の範囲に包含されることを理解すベ きである。 このような活性ドメインの好ましい例としては、 配列表の配列番号 1 に記載したァミノ酸配列の 2 6〜 3 9 8または配列番号 3に記載したァミノ酸配 列の 6 8〜3 9 8により特定される O-glycan α 2， 8 -シアル酸転移酵素の活性ド メインを挙げることができる。 また、 配列表の配列番号 1または配列番号 3に記 載したアミノ酸配列の 2 6〜1 0 0前後までの配列はステムと呼ばれる領域なの で活性には必ずしも必須ではないと考えられる。 従って、 配列表の配列番号 1ま たは配列番号 3に記載したァミノ酸配列の 1 0 1〜 3 9 8の領域を O- glycan a 2, 8 -シアル酸転移酵素の活性ドメインとして使用してもよい。 Furthermore, the active domain of the O-glycan α2,8-sialyltransferase of the present invention, or the O-glycan ct 2,8-sialyltransferase obtained by modifying or modifying a part of the amino acid sequence thereof It should be understood that all active proteins are included in the scope of the present invention. Preferred examples of such an active domain are specified by 26 to 398 of the amino acid sequence described in SEQ ID NO: 1 of the Sequence Listing or 68 to 398 of the amino acid sequence described in SEQ ID NO: 3 in the Sequence Listing. O-glycan α2,8-sialyltransferase activity domain. Also described in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing. The sequence of about 26 to 100 in the amino acid sequence described above is considered to be not necessarily essential for the activity since it is a region called a stem. Therefore, the region from 101 to 398 of the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing was used as the active domain of O-glycan a 2,8-sialyltransferase. Is also good.
即ち、本発明によれば、下記の何れかのァミノ酸配列を有する O -glycan α 2， 8- シアル酸転移酵素活性ドメィンから成る蛋白質が提供される。  That is, according to the present invention, there is provided a protein comprising an O-glycan α2,8-sialyltransferase active domain having any of the following amino acid sequences.
( 1 ) 配列表の配列番号 1に記載のァミノ酸配列のァミノ酸番号 2 6〜 3 9 8力 ら成るアミノ酸配列；  (1) an amino acid sequence consisting of amino acid numbers 26 to 398 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing;
( 2 ) 配列表の配列番号 1に記載のァミノ酸配列のァミノ酸番号 2 6〜 3 9 8力 ら成るアミノ酸配列において 1から数個のァミノ酸の欠失、 置換及び Ζ又は付加 を有するアミノ酸配列を有し、 O -glycan α 2, 8-シアル酸転移を触媒する活性を 有するアミノ酸配列：  (2) an amino acid having amino acid number 26 to 398 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, amino acid having deletion, substitution and 欠 失 or addition of one to several amino acids in the amino acid sequence An amino acid sequence having an activity of catalyzing O-glycan α2, 8-sialyltransferase:
( 3 ) 配列表の配列番号 3に記載のァミノ酸配列のァミノ酸番号 6 8〜 3 9 8力 ら成るアミノ酸配列；又は  (3) an amino acid sequence comprising amino acid numbers 68 to 398 of the amino acid sequence described in SEQ ID NO: 3 in the sequence listing; or
( 4 ) 配列表の配列番号 3に記載のァミノ酸配列のァミノ酸番号 6 8〜 3 9 8か ら成るアミノ酸配列において 1から数個のァミノ酸の欠失、 置換及び Ζ又は付加 を有するアミノ酸配列を有し、 O-glycan α 2, 8-シアル酸転移を触媒する活性を 有するアミノ酸配列：  (4) an amino acid having deletion, substitution, and Ζ or addition of one to several amino acids in the amino acid sequence consisting of amino acid numbers 68 to 398 of the amino acid sequence described in SEQ ID NO: 3 in the sequence listing An amino acid sequence having an activity of catalyzing O-glycan α 2, 8-sialyltransferase:
一方、 本発明の /3—ガラク トシド α 2， 6—シアル酸転移酵素は、 以下の作用 および基質特異性を有することを特徴とする。  On the other hand, the / 3-galactoside α2,6-sialyltransferase of the present invention is characterized by having the following action and substrate specificity.
( 1 ) 作用 ；  (1) action;
末端にガラクトース / 3 1， 4 Ν—ァセチルダルコサミン構造をもつ糖鎖のガラ ク トース部分に α 2 , 6の結合様式でシアル酸を転移する。  Sialic acid is transferred to the galactose part of the sugar chain having a galactose / 31,4-percetyldarcosamine structure at the terminal by α2,6 binding.
( 2 ) 基質特異性；  (2) substrate specificity;
末端にガラク ト一ス ]3 1 , 4 Ν—ァセチルダルコサミン構造をもつ糖鎖を基質 とし、 ラク トース、 及ぴ末端にガラク トース J3 1 , 3 N—ァセチルダルコサミン 構造をもつ糖鎖を基質としない。 上記した作用及び基質特異性性は、 本明細書に記載した実施例で取得されたヒ トおよびマウス由来の ]3—ガラク トシド α 2 , 6—シアル酸転移酵素について実 証された性質である。 本発明の —ガラク トシド α 2， 6—シアル酸転移酵素の 由来はヒ トまたはマウス由来のものに限定されるものではなく、 同型の ]3—ガラ クトシド α 2 , 6—シアル酸転移酵素が他の哺乳類の組織に存在し、 かつ、 それ らの ]3—ガラク トシド α 2 , 6—シアル酸転移酵素が互いに高度の相同性を有し ていることは当業者に容易に理解される。 [Galactose at the end] Lactose and a sugar chain having a galactose J31,3N-acetyldarcosamine structure at the terminal, using a sugar chain having a 31,4 1-acetylacetylcosamine structure as a substrate Is not used as a substrate. The actions and substrate specificities described above are properties demonstrated for the human and mouse-derived] 3-galactoside α2,6-sialyltransferase obtained in the examples described in the present specification. . The origin of the -galactoside α2,6-sialyltransferase of the present invention is not limited to that derived from human or mouse, and the same type of] -galactoside α2,6-sialyltransferase is used. It will be readily appreciated by those skilled in the art that the [3-galactoside α2,6-sialyltransferases present in other mammalian tissues and have a high degree of homology to one another.
このような ]3—ガラク トシド α 2 , 6—シアル酸転移酵素は、 上記した作用お よび基質特異性を有することを特徴とするものであり、 すべて本発明の範囲に属 するものである。  Such a] 3-galactoside α2,6-sialyltransferase is characterized by having the above-mentioned action and substrate specificity, and all belong to the scope of the present invention.
このような酵素としては、 哺乳類組織由来の天然型酵素やその変異体、 または β—ガラタトシド α 2 , 6—シアル酸転移を触媒し、 遺伝子組み換え技術により 製造された細胞外分泌型蛋白質などを挙げることができるが、 これらはいずれも 本発明の範囲に包含されるものである。  Examples of such enzymes include natural enzymes derived from mammalian tissues and mutants thereof, and extracellular secretory proteins produced by gene recombination technology that catalyze the transfer of β-galatatoside α2,6-sialyl acid. However, these are all included in the scope of the present invention.
本発明の ]3—ガラク トシド α 2 , 6—シアル酸転移酵素の一例としては、 下記 の何れかのアミノ酸配列を有する /3—ガラクトシド α 2 , 6—シアル酸転移酵素 が挙げられる。  An example of the [3-galactoside α2,6-sialyltransferase of the present invention] is a / 3-galactoside α2,6-sialyltransferase having any of the following amino acid sequences.
( 1 ) 配列表の配列番号 5または 7に記載のァミノ酸配列；又は  (1) an amino acid sequence according to SEQ ID NO: 5 or 7 in the sequence listing; or
( 2 ) 配列表の配列番号 5または 7に記載のァミノ酸配列において 1から数個の アミノ酸の欠失、 置換及ぴ 又は付加を有するアミノ酸配列を有し、 β —ガラク トシド α 2 , 6—シアル酸転移を触媒する活性を有するアミノ酸配列：  (2) an amino acid sequence represented by SEQ ID NO: 5 or 7 in the sequence listing, which has an amino acid sequence having deletion, substitution and / or addition of one to several amino acids, and β-galactoside α 2, 6- Amino acid sequence having activity for catalyzing sialyl transfer:
さらに、 本発明の ]3—ガラク トシド α 2 , 6—シアル酸転移酵素の活性ドメィ ン、 あるいはそのアミノ酸配列の一部を改変又は修飾して得られる/ 3—ガラク ト シド ct 2， 6—シアル酸転移酵素活性を有する蛋白質は全て本発明の範囲に包含 されることを理解すべきである。このような活性ドメィンの好ましい例としては、 配列表の配列番号 5に記載したアミノ酸配列の 3 3〜5 2 9にょり特定される]3 一ガラク トシド " 2， 6—シアル酸転移酵素の活性ドメインを挙げることができ る。 また、 配列表の配列番号 5に記載したァミノ酸配列の 3 1〜 2 0 0前後まで の配列はステムと呼ばれる領域なので活性には必ずしも必須ではないと考えられ る。 従って、 配列表の配列番号 1に記載したァミノ酸配列の 2 0 1〜 5 2 9の領 域を )3—ガラク トシド α 2 , 6—シアル酸転移酵素の活性ドメインとして使用し てもよい。 Furthermore, the [3-galactoside α2,6-sialyltransferase of the present invention] is obtained by modifying or modifying an active domain thereof or a part of the amino acid sequence thereof / 3-galactoside ct2,6- It should be understood that all proteins having sialyltransferase activity are included in the scope of the present invention. Preferred examples of such an active domain include those specified in 33 to 529 of the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing.] 3 Activity of monogalactoside "2,6-sialyltransferase Domain You. In addition, since the sequence of about 31 to 200 of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing is a region called a stem, it is considered that the sequence is not necessarily essential for activity. Therefore, the region from 201 to 5229 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing may be used as the active domain of) 3-galactoside α2,6-sialyltransferase.
同様に、 活性ドメインの好ましい例としては、 配列表の配列番号 7に記載した アミノ酸配列の 3 1〜5 2 4により特定される β—ガラタトシド α 2 , 6—シァ ル酸転移酵素の活性ドメィンを挙げることができる。 また、 配列表の配列番号 7 に記載したアミノ酸配列の 3 ：!〜 2 0 0前後までの配列はステムと呼ばれる領域 なので活性には必ずしも必須ではないと考えられる。 従って、 配列表の配列番号 7に記載したァミノ酸配列の 2 0 1〜 5 2 4の領域を /3 _ガラク トシド α 2 , 6 —シアル酸転移酵素の活性ドメインとして使用してもよい。  Similarly, as a preferred example of the active domain, an active domain of β-galatatoside α2,6-sialyltransferase identified by 31 to 524 of the amino acid sequence described in SEQ ID NO: 7 in the sequence listing may be used. Can be mentioned. In addition, since the sequence from 3 :! to around 200 in the amino acid sequence described in SEQ ID NO: 7 in the sequence listing is a region called a stem, it is considered that the sequence is not necessarily essential for the activity. Therefore, the region of 201 to 524 of the amino acid sequence described in SEQ ID NO: 7 in the sequence listing may be used as the active domain of / 3_galactoside α2,6-sialyltransferase.
即ち、 本発明によれば、 下記の何れかのアミノ酸配列を有する）3 _ガラク トシ ド α 2 , 6—シアル酸転移酵素活性ドメインから成る蛋白質が提供される。  That is, according to the present invention, there is provided a protein comprising any of the following amino acid sequences: 3_galactoside α2,6-sialyltransferase active domain.
本発明のさらに別の側面によれば、 下記の何れかのァミノ酸配列を有する /3 - ガラク トシド α 2， 6—シアル酸転移酵素活性ドメインから成る蛋白質が提供さ れる。  According to still another aspect of the present invention, there is provided a protein comprising a / 3-galactoside α2,6-sialyltransferase active domain having any one of the following amino acid sequences.
( 1 ) 配列表の配列番号 5に記載のアミノ酸配列のアミノ酸番号 3 3〜5 2 9力、 ら成るアミノ酸配列；  (1) an amino acid sequence consisting of amino acid numbers 33 to 529 of the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing;
( 2 ) 配列表の配列番号 5に記載のァミノ酸配列のァミノ酸番号 3 3〜 5 2 9カ ら成るアミノ酸配列において 1から数個のアミノ酸の欠失、 置換及び/又は付加 を有するアミノ酸配列を有し、 ）3—ガラク トシド α 2 , 6—シアル酸転移を触媒 する活性を有するアミノ酸配列：  (2) an amino acid sequence having deletion, substitution and / or addition of one to several amino acids in the amino acid sequence consisting of amino acids 33 to 529 of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing ) An amino acid sequence having the activity of catalyzing 3-galactoside α2,6-sialyltransferase:
( 3 ) 配列表の配列番号 7に記載のァミノ酸配列のァミノ酸番号 3 1〜 5 2 4カ ら成るアミノ酸配列；又は  (3) an amino acid sequence consisting of amino acid numbers 31 to 524 of the amino acid sequence described in SEQ ID NO: 7 in the sequence listing; or
( 4 ) 配列表の配列番号 7に記載のァミノ酸配列のァミノ酸番号 3 1〜 5 2 4か ら成るアミノ酸配列において 1から数個のアミノ酸の欠失、 置換及び Ζ又は付加 を有するアミノ酸配列を有し、 一ガラクトシド " 2 , 6—シアル酸転移を触媒 する活性を有するアミノ酸配列： (4) Deletion, substitution and Ζ or addition of one to several amino acids in the amino acid sequence consisting of amino acid numbers 31 to 524 of the amino acid sequence described in SEQ ID NO: 7 in the sequence listing An amino acid sequence having the activity of catalyzing the transfer of one galactoside "2,6-sialyl acid:
本明細書で言う 「1から数個のアミノ酸の欠失、 置換及び Z又は付加を有する アミノ酸配列」 における 「1から数個」 の範囲は特には限定されないが、 例えば、 1から 2 0個、 好ましくは 1から 1 0個、 より好ましくは 1から 7個、 さらに好 ましくは 1から 5個、 特に好ましくは 1から 3個程度を意味する。  The range of `` 1 to several '' in the `` amino acid sequence having deletion, substitution and Z or addition of 1 to several amino acids '' referred to herein is not particularly limited, for example, 1 to 20 It preferably means about 1 to 10, more preferably about 1 to 7, even more preferably about 1 to 5, particularly preferably about 1 to 3.
本発明の酵素又は蛋白質の取得方法については特に制限はなく、 化学合成によ り合成した蛋白質でもよいし、 遺伝子組み換え技術により作製した組み換え蛋白 質でもよい。  The method for obtaining the enzyme or protein of the present invention is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by a genetic recombination technique.
組み換え蛋白質を作製する場合には、 先ず当該蛋白質をコードする D N Aを入 手することが必要である。 本明細書の配列表の配列番号 1から 8に記載したァミ ノ酸配列および塩基配列の情報を利用することにより適当なプライマーを設計 し、 それらを用いて適当な c D N Aライブラリーを铸型にして P C Rを行うこと により、 本発明の酵素をコードする D N Aを取得することができる。  When producing a recombinant protein, it is necessary to first obtain DNA encoding the protein. Using the amino acid sequence and base sequence information described in SEQ ID NOs: 1 to 8 in the sequence listing of the present specification, appropriate primers are designed, and using them, an appropriate cDNA library is formed. By performing PCR in this manner, a DNA encoding the enzyme of the present invention can be obtained.
例えば、 配列番号 1および配列番号 3に記載のァミノ酸配列を有する O -glycan α 2, 8-シアル酸転移酵素をコードする cDNA、 並びに配列番号 5および配 列番号 7に記載のアミノ酸配列を有する 一ガラクトシド α 2 , 6—シアル酸転 移酵素をコードする cDNA を単離する方法は以下の実施例に詳細に説明されてい る。 もっとも、 本発明の O- glycan α 2， 8-シアル酸転移酵素または ]3—ガラタ ト シド α 2 , 6—シアル酸転移酵素をコードする cDNAの単離方法はこれらの方法に 限定されることはなく、 当業者は下記の実施例に記載された方法を参照しつつ、 この方法を適宜修飾ないし変更することにより、容易に目的の cDNAを単離するこ とができる。  For example, a cDNA encoding O-glycan α2,8-sialyltransferase having the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 7 Methods for isolating cDNA encoding monogalactoside α2,6-sialyltransferase are described in detail in the Examples below. However, the method for isolating the cDNA encoding the O-glycan α2,8-sialyltransferase or] 3-galatatoside α2,6-sialyltransferase of the present invention is limited to these methods. However, those skilled in the art can easily isolate the desired cDNA by appropriately modifying or changing this method while referring to the method described in the following Examples.
また、 本発明の酵素をコ一ドする D N Aの一部の断片を上記した P C Rにより 得た場合には、 作製した D N A断片を順番に遺伝子組み換え技術により連結する ことにより、 所望の酵素をコードする D N Aを得ることができる。 この D N Aを 適当な発現系に導入することにより、 本発明の酵素を産生することができる。 発 現系での発現については本明細書中後記する。 When a partial fragment of DNA encoding the enzyme of the present invention is obtained by the above-described PCR, the produced DNA fragments are sequentially ligated by gene recombination to encode a desired enzyme. DNA can be obtained. The enzyme of the present invention can be produced by introducing this DNA into an appropriate expression system. Departure The expression in the current system will be described later in this specification.
さらに、 本発明の 0 - glycan α 2, 8-シアル酸転移酵素または —ガラク トシド α 2 , 6—シアル酸転移酵素の活性ドメインであるポリペプチド部分とシグナル ペプチドとを含む細胞外分泌型の蛋白であって、 O - glycan α 2，8-シアル酸転移 または β —ガラク トシド α 2 , 6—シアル酸転移を触媒する活性を有する蛋白質 も本発明に含まれる。  Further, the present invention relates to an extracellular secretory protein comprising a polypeptide portion which is an active domain of 0-glycan α2,8-sialyltransferase or -galactoside α2,6-sialyltransferase of the present invention and a signal peptide. In addition, a protein having an activity of catalyzing O-glycan α2,8-sialyltransfer or β-galactoside α2,6-sialyltransfer is also included in the present invention.
本発明の 0- glycan α 2, 8-シアル酸転移酵素おょぴ ]3—ガラク トシド α 2 , 6 ーシアル酸転移酵素は、 発現後に細胞内に留まり、 細胞外に分泌されない場合が ある。 また、 細胞内濃度が一定以上になると、 酵素の発現量が低下するという可 能性がある。 上記の Ο - glycan α 2, 8-シアル酸転移酵素の O- glycan ct 2, 8-シァ ル酸転移活性および J3 _ガラク トシド α 2 , 6—シアル酸転移酵素の 一ガラク トシド^ 2 , 6—シアル酸転移活性を有効に利用するために、 本酵素の活性を維 持し、 かつ発現時に細胞から分泌される可溶性形態の蛋白を製造することができ る。 このような蛋白としては、 本発明の 0 - glycan α 2, 8_シアル酸転移酵素また は /3 —ガラク トシド α 2， 6—シアル酸転移酵素の活性に関与する O-glycan α 2， 8-シアル酸転移酵素または /3—ガラクトシドひ 2， 6—シアル酸転移酵素の活 性ドメインであるポリぺプチド部分とシグナルぺプチドとを含む細胞外分泌型の 蛋白であって、 0 - glycan α 2, 8-シアル酸転移または j3 _ガラク トシド α 2 , 6 —シアル酸転移を触媒する蛋白質を挙げることができる。 例えば、 マウス免疫グ ロブリン IgMのシグナルペプチドや、 プロテイン Aとの融合蛋白は本発明の分泌 型蛋白の好ましい態様である。 The 0-glycan α2,8-sialyltransferase of the present invention] -galactoside α2,6-sialyltransferase may remain in the cell after expression and may not be secreted out of the cell. In addition, when the intracellular concentration exceeds a certain level, the expression level of the enzyme may decrease. Additional Ο - glycan α 2, 8- sialyltransferase of O-Glycan ct 2, 8- Xia Le acid transferase activity, and J3 _ galactosyl Toshido alpha 2, 6- one galactokinase sialyltransferase Toshido ^ 2, 6 —To make effective use of the sialyltransferase activity, it is possible to maintain the activity of the enzyme and produce a soluble form of the protein that is secreted from cells during expression. Such proteins include the 0-glycan α2,8_sialyltransferase or the O-glycan α2,8 involved in the activity of / 3-galactoside α2,6-sialyltransferase of the present invention. 0-glycan α2 is an extracellular secretory protein containing the polypeptide portion and the signal peptide, which are the active domain of -sialyltransferase or / 3-galactoside 2,6-sialyltransferase. , 8-sialyltransfer or j3_galactoside α 2,6 -sialyltransfer. For example, a signal peptide of mouse immunoglobulin IgM and a fusion protein with protein A are preferred embodiments of the secretory protein of the present invention.
これまでにクローユングされたシアル酸転移酵素は、 他のダリコシルトランス フェラーゼと同様のドメイン構造を有している。 すなわち、 NH2末端の短い細胞 質中尾部、 疎水性のシグナルアンカードメイン、 蛋白分解感受性を有するステム (stem)領域、 及ぴ C00H-末端の大きな活性ドメインを有する（Paulson, J. C. and Col ley, K. J. , J. Biol. Chew. , 264, 17615-17618, 1989)。 本発明の O - glycan a 2, 8-シアル酸転移酵素または ;3—ガラク トシド a 2， 6—シアル酸転移酵素の 経膜ドメインの位置を調べるためには、 カイ ト及びドゥーリ トル （Kyte， J. and Dool ittle, R. F. , J. Mol. Biol. , 157, 105-132, 1982) の方法に従って作成した 疎水性分布図を利用することができる。 また、 活性ドメイン部分の推定には、 各 種のフラグメンドを導入した組換えプラスミ ドを作成して利用することができ る。 このような方法の一例は、例えば PCT/JP94/02182号の明細書に詳細に記載さ れているが、 経膜ドメインの位置の確認や活性ドメイン部分の推定方法は、 この 方法に限定されることはない。 So far, sialyltransferases that have been clawed have a domain structure similar to that of other dalycosyltransferases. That, NH 2 terminal short cytoplasmic Nakao unit, hydrophobic signal anchor domain, a stem (stem) regions with proteolytic susceptibility, has a large active domain of及Pi C00H- end (Paulson, JC and Col ley, KJ , J. Biol. Chew., 264, 17615-17618, 1989). The O-glycan a 2,8-sialyltransferase or the 3-galactoside a 2,6-sialyltransferase of the present invention; To determine the location of the transmembrane domain, a hydrophobic distribution prepared according to the method of Kite and Doolittle (RF, J. Mol. Biol., 157, 105-132, 1982) was used. Figures are available. In addition, for estimating the active domain portion, a recombinant plasmid into which various types of fragments are introduced can be used. One example of such a method is described in detail in, for example, the specification of PCT / JP94 / 02182, but the method of confirming the location of the transmembrane domain and estimating the active domain portion is limited to this method. Never.
O-glycan α 2, 8-シアル酸転移酵素または ]3 —ガラクトシド α 2 , 6—シアル 酸転移酵素の活性ドメインであるポリぺプチド部分とシグナルぺプチドとを含む 細胞外分泌型の蛋白の製造のためには、 例えばシグナルぺプチドとして免疫グロ ブリンシグナルペプチド配列を用い、 O- glycan ひ 2， 8_シアル酸転移酵素または ]3—ガラク トシド α 2 , 6—シアル酸転移酵素の活性ドメインに対応する配列を 該シグナルぺプチドにインフレーム融合させればょレ、。このような方法としては、 例えば、 ジョブリンの方法（Jobling, S. A. and Gehrke, L. , Nature (Ιοηά. ) , 325, 622-625, 1987) を利用することができる。 また、 本明細書の実施例に詳細に説明 されているように、 マウス免疫グロブリン IgMのシグナルペプチドやプロテイン Aとの融合蛋白を製造してもよい。 もっとも、 シグナルペプチドの種類やシグナ ルぺプチドと活性ドメインの結合方法、 または可溶化の方法は上記方法に限定さ れることはなく、 当業者は、 O-glycan α 2, 8-シアル酸転移酵素または 3—ガラ ク トシド α 2， 6—シアル酸転移酵素の活性ドメインであるポリペプチド部分を 適宜選択することができるし、 それらを利用可能な任意のシグナルべプチドと適 宜の方法により結合することにより細胞外分泌型の蛋白を製造することができ る。  O-glycan α 2, 8-sialyltransferase or] 3-galactoside α2,6-sialyltransferase The production of extracellular secretory proteins containing the polypeptide part of the active domain and the signal peptide For example, using the immunoglobulin signal peptide sequence as the signal peptide, it corresponds to the active domain of O-glycan 2,8-sialyltransferase or] 3-galactoside α2,6-sialyltransferase A sequence to be fused to the signal peptide in frame. As such a method, for example, Joblin's method (Jobling, S.A. and Gehrke, L., Nature (Ιοηά.), 325, 622-625, 1987) can be used. Further, as described in detail in the examples of the present specification, a fusion protein with mouse A immunoglobulin IgM signal peptide or protein A may be produced. However, the type of signal peptide, the method of binding the signal peptide to the active domain, or the method of solubilization are not limited to the above methods, and those skilled in the art may use O-glycan α2, 8-sialyltransferase. Alternatively, the polypeptide moiety that is the active domain of 3-galactoside α2,6-sialyltransferase can be selected as appropriate, and they can be bound to any available signal peptide by an appropriate method. As a result, an extracellular secretory protein can be produced.
( 2 ) 本発明の遺伝子 (2) Gene of the present invention
本発明によれば、 本発明の 0- glycan α 2, 8 -シアル酸転移酵素のアミノ酸配列 をコードする遺伝子、 並びに] 3 _ガラク トシド α 2 , 6—シアル酸転移酵素のァ ミノ酸配列をコードする遺伝子が提供される。 According to the present invention, a gene encoding the amino acid sequence of 0-glycan α2,8-sialyltransferase of the present invention, and a gene encoding 3_galactoside α2,6-sialyltransferase A gene encoding a amino acid sequence is provided.
本発明の O- glycan α 2， 8_シアル酸転移酵素のァミノ酸配列をコードする遺伝 子の具体例としては、 下記の何れかの塩基配列を有する遺伝子が挙げられる。  Specific examples of the gene encoding the amino acid sequence of the O-glycan α2,8_sialyltransferase of the present invention include a gene having any one of the following nucleotide sequences.
(1) 配列表の配列番号 2に記載の塩基配列中の塩基番号 7 7番目から 1 2 70 番目で特定される塩基配列；  (1) a nucleotide sequence identified by nucleotide numbers 77 to 127 in the nucleotide sequence of SEQ ID NO: 2 in the sequence listing;
(2) 配列表の配列番号 2に記載の塩基配列中の塩基番号 7 7番目から 1 2 70 番目で特定される塩基配列において 1から数個の塩基の欠失、 置換及び Ζ又は付 加を有する塩基配列を有し、 O-glycan ct 2, 8-シアル酸転移を触媒する活性を有 する蛋白質をコードする塩基配列：  (2) Deletion, substitution and Ζ or addition of one to several bases in the base sequence specified by base numbers 77 to 127 in the base sequence described in SEQ ID NO: 2 in the sequence listing A nucleotide sequence encoding a protein having an activity of catalyzing O-glycanct 2,8-sialyltransferase having the following nucleotide sequence:
(3) 配列表の配列番号 4に記載の塩基配列中の塩基番号 9 2番目から 1 28 5 番目で特定される塩基配列；  (3) a base sequence specified by base numbers 9 from 2 to 1285 in the base sequence of SEQ ID NO: 4 in the sequence listing;
(4) 配列表の配列番号 4に記載の塩基配列中の塩基番号 9 2番目から 1 28 5 番目で特定される塩基配列において 1から数個の塩基の欠失、 置換及び 又は付 加を有する塩基配列を有し、 O-glycan α 2, 8-シアル酸転移を触媒する活性を有 する蛋白質をコードする塩基配列：  (4) In the nucleotide sequence set forth in SEQ ID NO: 4 in the nucleotide sequence listed in SEQ ID NO: 4 there is a deletion, substitution, and / or addition of one to several nucleotides in the nucleotide sequence specified by nucleotide numbers 9 to 1285 A nucleotide sequence encoding a protein having a nucleotide sequence and having an activity of catalyzing O-glycan α2,8-sialyltransferase:
本発明の ]3—ガラク トシド α 2， 6—シアル酸転移酵素のアミノ酸配列をコー ドする遺伝子の具体例としては、 下記の何れかの塩基配列を有する遺伝子が挙げ られる。  Specific examples of the gene encoding the amino acid sequence of the 3-galactoside α2,6-sialyltransferase of the present invention include a gene having any one of the following nucleotide sequences.
(1) 配列表の配列番号 6に記載の塩基配列中の塩基番号 1 7 6番目から 1 76 2番目で特定される塩基配列；  (1) a nucleotide sequence specified by nucleotide numbers 176 to 176 in the nucleotide sequence of SEQ ID NO: 6 in the sequence listing;
(2) 配列表の配列番号 6に記載の塩基配列中の塩基番号 1 7 6番目から 1 76 2番目で特定される塩基配列において 1から数個の塩基の欠失、 置換及び/又は 付加を有する塩基配列を有し、 —ガラクトシド《 2， 6—シアル酸転移を触媒 する活性を有する蛋白質をコードする塩基配列：  (2) deletion, substitution and / or addition of one to several bases in the base sequence specified by base numbers 176 to 176 in the base sequence described in SEQ ID NO: 6 in the sequence listing A nucleotide sequence encoding a protein having an activity of catalyzing transfer of -galactoside << 2,6-sialyl acid:
(3) 配列表の配列番号 8に記載の塩基配列中の塩基番号 3番目から 1 5 74番 目で特定される塩基配列；又は  (3) a nucleotide sequence specified by nucleotides 3 through 1574 in the nucleotide sequence of SEQ ID NO: 8 in the sequence listing; or
(4) 配列表の配列番号 8に記載の塩基配列中の塩基番号 3番目から 1 5 74番 目で特定される塩基配列において 1から数個の塩基の欠失、 置換及び 又は付加 を有する塩基配列を有し、 ）3 _ガラク トシド ct 2， 6 —シアル酸転移を触媒する 活性を有する蛋白質をコードする塩基配列： (4) 3rd to 1574th nucleotides in the nucleotide sequence of SEQ ID NO: 8 in the sequence listing A protein having a base sequence having deletion, substitution and / or addition of one to several bases in the base sequence specified by the eye, and having an activity of catalyzing transfer of 3 _ galactoside ct 2, 6-sialic acid Nucleotide sequence encoding:
本明細書で言う 「1から数個の塩基の欠失、 置換及び Z又は付加を有する塩基 配列」 における 「1から数個」 の範囲は特には限定されないが、 例えば、 1から 6 0個、 好ましくは 1から 3 0個、 より好ましくは 1から 2 0個、 さらに好まし くは 1から 1 0個、 さらに好ましくは 1から 5個、 特に好ましくは 1から 3個程 度を意味する。  The range of `` 1 to several '' in the `` base sequence having 1 to several bases of deletion, substitution and Z or addition '' referred to herein is not particularly limited, for example, 1 to 60, Preferably about 1 to 30, more preferably about 1 to 20, more preferably about 1 to 10, more preferably about 1 to 5, particularly preferably about 1 to 3.
さらに、 本発明の O-glycan α 2， 8-シアル酸転移酵素または j3 —ガラク トシド a 2 , 6—シアル酸転移酵素の活性ドメインから成る蛋白質、 並びに該活性ドメ ィンであるポリぺプチド部分とシグナルぺプチドとを含む細胞外分泌型の蛋白で あって、 O-glycan α 2, 8-シアル酸転移または /3 —ガラクトシド α 2 , 6—シァ ル酸転移を触媒する活性を有する蛋白質をコードする遺伝子も本発明の範囲に属 する。  Further, a protein comprising an active domain of the O-glycan α2,8-sialyltransferase or j3-galactoside a2,6-sialyltransferase of the present invention, and a polypeptide moiety which is the active domain An extracellular secretory protein containing a protein and a signal peptide, which encodes a protein having an activity of catalyzing O-glycan α2,8-sialyltransfer or / 3-galactoside α2,6-sialyltransferase Genes belonging to the present invention also belong to the scope of the present invention.
本発明の遺伝子の取得方法は上述した通りである。  The method for obtaining the gene of the present invention is as described above.
また、 所定の核酸配列に所望の変異を導入する方法は当業者に公知である。 例 えば、 部位特異的変異誘発法、 縮重オリゴヌクレオチドを用いる P C R、 核酸を 含む細胞の変異誘発剤又は放射線への露出等の公知の技術を適宜使用することに よって、変異を有する D N Aを構築することができる。 このような公知の技術は、 例えは、 Molecular Cloning : A laboratory annual, 2nd Ed.， Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. , 1989、 並びに Current Protocols in Molecular Biology, Supplement 1〜38, John Wi ley & Sons (1987-1997)に記載 されている。 In addition, a method for introducing a desired mutation into a predetermined nucleic acid sequence is known to those skilled in the art. For example, a DNA having a mutation is constructed by appropriately using known techniques such as site-directed mutagenesis, PCR using a degenerate oligonucleotide, exposure of a cell containing nucleic acid to a mutagen or radiation. can do. Such known techniques, the example, Molecular Cloning:.. A laboratory annual, 2 nd Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, and Current Protocols in Molecular Biology, Supplement 1~38 , John Wiley & Sons (1987-1997).
( 3 ) 本発明の組み換えベクター (3) The recombinant vector of the present invention
本発明の遺伝子は適当なベクター中に挿入して使用することができる。 本発明 で用いるベクターの種類は特に限定されず、 例えば、 自立的に複製するベクター (例えばプラスミ ド等） でもよいし、 あるいは、 宿主細胞に導入された際に宿主 細胞のゲノムに組み込まれ、 組み込まれた染色体と共に複製されるものであって もよい。 The gene of the present invention can be used by inserting it into an appropriate vector. The type of vector used in the present invention is not particularly limited. For example, an autonomously replicating vector (Eg, plasmid), or it may be integrated into the genome of the host cell when introduced into the host cell and replicated along with the integrated chromosome.
好ましくは、 本発明で用いるベクターは発現ベクターである。 発現ベクターに おいて本発明の遺伝子は、 転写に必要な要素 （例えば、 プロモーター等） が機能 的に連結されている。 プロモータは宿主細胞において転写活性を示す D N A配列 であり、 宿主の種類に応じて適宜選択することができる。  Preferably, the vector used in the present invention is an expression vector. In the expression vector, the gene of the present invention is operably linked to elements required for transcription (for example, a promoter and the like). The promoter is a DNA sequence showing transcription activity in a host cell, and can be appropriately selected depending on the type of the host.
細菌細胞で作動可能なプロモータとしては、 バチルス · ステア口テルモフィル ス · マノレ ト シエニック · ア ^ フ一で遣伝子 (Bacillus stearothermophi丄 us maltogenic amylase gene)、 /ヽチノレス · リケニホノレ^ス a , フ'— 遺 十 (Baci llus l icheniformis alpha— amylase gene)、 ノ テノレス 'ア^ロリケフアチェ ンス · BA ァミフ— ^遺 1 子 (Baci l lus amyloliquefaciens BAN amylase gene)、 バチルス · サブチリス · アル力リプロテアーゼ遺伝子（Baci llus S ubti lis alkal ine protease gene)もしくはバチノレス ·プミノレス ·キシロシダーゼ遺伝子 (Bacil lus pumilus xylosldase gene)のプロモータ、 またはファージ · ラムダの P R若しくは 1\プロモータ、 大腸菌の lac、 trp若しくは tacプロモータなどが 挙げられる。 The promoters operable in bacterial cells include Bacillus stearothermophius us maltogenic amylase gene, Bacillus stearothermophius us maltogenic amylase gene, and Bacillus stearothermophius us maltogenic amylase gene. '—The ten (Baci llus l icheniformis alpha—amylase gene), not tenores' A ^^ orikef Achens · BA Amihu – ^ one child (Baci l lus amyloliquefaciens BAN amylase gene), the Bacillus subtilis · al force reprotease gene Baci llus S ubti lis alkal ine protease gene) or promoters Bachinoresu-Puminoresu-xylosidase gene (Bacil lus pumilus xylosldase gene) or phage lambda P R or 1 \ promoter, E. coli lac, etc. can be mentioned trp or tac promoter Can be
哺乳動物細胞で作動可能なプロモータの例としては、 S V 4 0プロモータ、 M T - 1 (メタ口チォネイン遺伝子） プロモータ、 またはアデノウイルス 2主後期 プロモータなどがある。 昆虫細胞で作動可能なプロモータの例としては、 ポリへ ドリンプロモータ、 P 1 0プロモータ、 ォートグラファ ' カリホル二力 ·ポリへ ドロシス塩基性タンパクプロモータ、 バキユウロウィルス即時型初期遺伝子 1プ 口モータ、 またはバキユウロウィルス 3 9 K遅延型初期遺伝子プロモータ等があ る。 酵母宿主細胞で作動可能なプロモータの例としては、 酵母解糖系遺伝子由来 のプロモータ、 アルコールデヒ ドロゲナーゼ遺伝子プロモータ、 T P I 1プロモ ータ、 A D H2-4Cプロモータなどが挙げられる。  Examples of promoters operable in mammalian cells include the SV40 promoter, the MT-1 (metamouth thionein gene) promoter, or the adenovirus 2 major late promoter. Examples of promoters operable in insect cells include the polyhedrin promoter, the P10 promoter, the autographer's californica-polyhedrosis basic protein promoter, the baculourovirus immediate-early gene 1-port motor, and the baki Eurovirus 39K There is a delayed-type early gene promoter. Examples of promoters operable in yeast host cells include promoters derived from yeast glycolysis genes, alcohol dehydrogenase gene promoters, TPI1 promoters, ADH2-4C promoters, and the like.
糸状菌細胞で作動可能なプロモータの例としては、 A D H 3プロモータまたは t p i Aプロモータなどがある。 Examples of promoters operable in filamentous fungal cells include the ADH3 promoter or There is a tpi A promoter.
また、 本発明の DNAは必要に応じて、 例えばヒ ト成長ホルモンターミネータ または真菌宿主については TP I 1ターミネータ若しくは ADH3ターミネータ のような適切なターミネータに機能的に結合されてもよい。 本発明の組み換えべ クタ一は更に、ポリアデニレーシヨンシグナル（例えば SV40またはアデノウイ ルス 5 E 1 b領域由来のもの）、転写ェンハンサ配列（例えば SV 40ェンハンサ） および翻訳ェンハンサ配列（例えばアデノウイルス VA RNA をコードするも の） のような要素を有していてもよい。  The DNA of the present invention may also be operably linked to a suitable terminator, such as, for example, a human growth hormone terminator or, for fungal hosts, a TPI1 terminator or an ADH3 terminator. The recombinant vector of the present invention further comprises a polyadenylation signal (eg, from the SV40 or adenovirus 5E1b region), a transcription enhancer sequence (eg, the SV40 enhancer) and a translation enhancer sequence (eg, the adenovirus VA RNA). May be included.
本発明の組み換えべクタ一は更に、 該べクターが宿主細胞内で複製することを 可能にする DN A配列を具備してもよく、その一例としては SV 40複製起点（宿 主細胞が哺乳類細胞のとき） が挙げられる。  The recombinant vector of the present invention may further comprise a DNA sequence that allows the vector to replicate in a host cell, such as the SV40 origin of replication (where the host cell is a mammalian cell). At the time of).
本発明の組み換えベクターはさらに選択マーカーを含有してもよい。 選択マ一 カーとしては、 例えば、 ジヒ ドロ葉酸レダクターゼ （DHFR) またはシゾサッ カロマイセス ·ボンべ TP I遺伝子等のようなその補体が宿主細胞に欠けている 遺伝子、 または例えばアンピシリン、 カナマイシン、 テトラサイクリン、 クロラ ムフエ二コール、 ネオマイシン若しくはヒグロマイシンのような薬剤耐性遺伝子 を挙げることができる。  The recombinant vector of the present invention may further contain a selection marker. Selection markers include, for example, genes whose complement is lacking in the host cell, such as dihydrofolate reductase (DHFR) or the Schizosaccharomyces bombi TPI gene, or ampicillin, kanamycin, tetracycline, chlora Drug resistance genes such as mufenicol, neomycin or hygromycin can be mentioned.
本発明の DNA、 プロモータ、 および所望によりターミネータおよびノまたは 分泌シグナル配列をそれぞれ連結し、 これらを適切なベクターに挿入する方法は 当業者に周知である。  Methods of ligating the DNA, promoter, and, if desired, terminator and no or secretory signal sequence of the present invention, respectively, and inserting these into an appropriate vector are well known to those skilled in the art.
(4) 本発明の形質転換体及びそれを用いた蛋白質の製造 (4) Transformant of the present invention and production of protein using the same
本発明の DN A又は組み換えベクターを適当な宿主に導入することによって形 質転換体を作製することができる。  Transformants can be prepared by introducing the DNA or recombinant vector of the present invention into a suitable host.
本発明の DNAまたは組み換えベクターを導入される宿主細胞は、 本発明の D NA構築物を発現できれば任意の細胞でよく、 細菌、 酵母、 真菌および高等真核 細胞等が挙げられる。 細菌細胞の例としては、 バチルスまたはストレプトマイセス等のグラム陽性菌 又は大腸菌等のグラム陰性菌が挙げられる。 これら細菌の形質転換は、 プロ トプ ラスト法、または公知の方法でコンビテント細胞を用いることにより行えばよレ、。 哺乳類細胞の例としては、 H E K 2 9 3細胞、 H e L a細胞、 C O S細胞、 B H K細胞、 C H L細胞または C HO細胞等が挙げられる。 哺乳類細胞を形質転換 し、 該細胞に導入された D N A配列を発現させる方法も公知であり、 例えば、 ェ レクト口ポーレーシヨン法、 リン酸カルシウム法、 リポフエクシヨン法等を用い ることができる。 The host cell into which the DNA or recombinant vector of the present invention is introduced may be any cell as long as it can express the DNA construct of the present invention, and includes bacteria, yeast, fungi, and higher eukaryotic cells. Examples of bacterial cells include Gram-positive bacteria such as Bacillus or Streptomyces or Gram-negative bacteria such as Escherichia coli. Transformation of these bacteria may be carried out by protoplast method or by using a competent cell by a known method. Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells or CHO cells. Methods for transforming a mammalian cell and expressing the DNA sequence introduced into the cell are also known. For example, an electoral port method, a calcium phosphate method, a lipofection method and the like can be used.
酵母細胞の例としては、 サッカロマイセスまたはシゾサッカロマイセスに属す る細胞が挙げられ、 例えば、 サッカロマイセス ' セレピシェ（Saccharomyces cerevislae) た fまサッカロマづセス · クノレイベリ、 S accharomyces kluyveri)等 が挙げられる。 酵母宿主への組み換えベクターの導入方法としては、 例えば、 ェ レク ト口ポレーシヨン法、 スフエロブラスト法、 酢酸リチウム法等を挙げること ができる。  Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, such as Saccharomyces cerevislae and Saccharomyces kluyveri. Examples of a method for introducing a recombinant vector into a yeast host include an electoral poration method, a spheroblast method, and a lithium acetate method.
他の真菌細胞の例は、 糸状菌、 例えばァスペルギルス、 ニューロスポラ、 フザ リゥム、 またはトリコデルマに属する細胞である。 宿主細胞として糸状菌を用い る場合、 D N A構築物を宿主染色体に組み込んで組換え宿主細胞を得ることによ り形質転換を行うことができる。 D N A構築物の宿主染色体への組み込みは、 公 知の方法に従い、 例えば相同組換えまたは異種組換えにより行うことができる。 昆虫細胞を宿主として用いる場合には、 組換え遺伝子導入ベクターおよびバキ ュロウィルスを昆虫細胞に共導入して昆虫細胞培養上清中に組換えウィルスを得 た後、 さらに組換えウィルスを昆虫細胞に感染させ、 蛋白質を発現させることが できる (例 ば、 Baculovirus Expression Vectors, A Laboratory Manual；及び カレント 'プロ トコールズ'イン'モレキュラー.バイオロジー、 Bio/Technology， 6, 47 (1988)等に記載) 。  Examples of other fungal cells are filamentous fungi, such as cells belonging to Aspergillus, Neurospora, Fusarium, or Trichoderma. When a filamentous fungus is used as a host cell, transformation can be performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be performed according to known methods, for example, by homologous recombination or heterologous recombination. When an insect cell is used as a host, the recombinant gene transfer vector and baculovirus are co-transfected into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is transmitted to the insect cells. Then, the protein can be expressed (for example, described in Baculovirus Expression Vectors, A Laboratory Manual; and current “Protocols” in “Molecular. Biology, Bio / Technology, 6, 47 (1988)).
バキュロウィルスとしては、 例えば、 ョ トウガ科昆虫に感染するウィルスであ るアウ トグラファ *カリフォルニ力 ·ヌクレア一 ·ポリへドロシス · ウィルス (Autographs californica nuclear polyhedrosis virus)等を用レヽることカ できる。 昆虫細胞としては、 Spodoptera frugiperda の卵巣細胞である S f 9、 S f 2 1 レ キュロウイノレス 'エクスプレッション 'ベクターズ、 ァ 'ラボラトリー ' マニュアル、ダブリユー'エイチ 'フリーマン 'アンド 'カンパ二一（W. H. Freeman and Company) , ニューヨーク（New York)、 （1992)〕 、 Trichoplusia ni の卵巣細 胞である H i F i v e (インビトロジェン社製）等を用いることができる。 Baculoviruses include, for example, Autographa, a virus that infects insects of the family Capitaridae * Californi, nuclei, polyhedrosis, virus (Autographs californica nuclear polyhedrosis virus) and the like. Insect cells include the ovary cells of Spodoptera frugiperda, Sf9, Sf21 Recuroinoles 'Expression' Vectors, A 'Laboratory' Manual, Double H 'Freeman' and 'Company' (WH Freeman and Company) ), New York, (1992)], and Trichoplusia ni ovarian cells, Hi Five (Invitrogen), and the like.
組換えウィルスを調製するための、 昆虫細胞への組換え遺伝子導入ベクターと 上記バキュロウィルスの共導入方法としては、 例えば、 リン酸カルシウム法又は リポフエクション法等を挙げることができる。  Examples of a method for co-introducing a recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus include a calcium phosphate method and a lipofection method.
上記の形質転換体は、 導入された D N A構築物の発現を可能にする条件下で適 切な栄養培地中で培養する。 形質転換体の培養物から、 本発明の酵素を単離精製 するには、 通常の蛋白質の単離、 精製法を用いればよい。  The above transformants are cultured in a suitable nutrient medium under conditions that allow expression of the introduced DNA construct. In order to isolate and purify the enzyme of the present invention from the culture of the transformant, a conventional protein isolation and purification method may be used.
例えば、本発明の酵素が、 細胞内に溶解状態で発現した場合には、培養終了後、 細胞を遠心分離により回収し水系緩衝液に懸濁後、 超音波破砕機等により細胞を 破砕し、 無細胞抽出液を得る。 該無細胞抽出液を遠心分離することにより得られ た上清から、 通常の蛋白質の単離精製法、 即ち、 溶媒抽出法、 硫安等による塩析 法、 脱塩法、 有機溶媒による沈殿法、 ジェチルアミノエチル (DEAE)セファロース 等のレジンを用いた陰イオン交換クロマトグラフィ一法、 S- Sepharose FF (フアル マシア社製)等のレジンを用いた陽イオン交換クロマトグラフィ一法、ブチルセフ ァロース、 フエ-ノレセファロース等のレジンを用いた疎水性ク口マトグラフィー 法、 分子篩を用いたゲルろ過法、 ァフィ二ティークロマトグラフィー法、 クロマ トフオーカシング法、 等電点電気泳動等の電気泳動法等の手法を単独あるいは組 み合わせて用い、 精製標品を得ることができる。  For example, when the enzyme of the present invention is expressed in a dissolved state in the cells, after the culture is completed, the cells are collected by centrifugation, suspended in an aqueous buffer, and then disrupted by an ultrasonic disrupter or the like. Obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal protein isolation and purification method, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as getylaminoethyl (DEAE) Sepharose, cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia), butyl sepharose, phenol Hydrophobic chromatography using a resin such as Sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, etc. Used in combination, a purified sample can be obtained.
以下の実施例により本発明をさらに具体的に説明するが、 本発明は実施例によ つて限定されるものではない。 実施例 実施例 1 : O - glycan α 2， 8-シアル酸転移酵素 The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples. Example Example 1: O-glycan α 2, 8-sialyltransferase
本発明の具体例に用いた試薬、 試料類は以下の通りである。 Fetuin, asialofetuin, bovine submaxi llary mucin (BSM) , a 1 - acid glycoprotein, ovomucoid, lactosyl ceramide (LacCer) , GM3, GMla, GDI a, GDlb, iiTlb, CMP-NeuAc, 6' -sialyllactose, 3' -sialyl-vV-acetyllactosamine, Triton CF-54 は Sigma 社力 ら購入した。 3'—sialyllactose, 6'— sialyl-jV— acetyllactosamine は Calbiochem社から購入した。 -ァセチルノイラミン酸（NeuAc) , G 4, Gal, N- ァセチルガラクトサミン（GalNAc)は和光純薬から購入した。 GD3 は雪印乳業から 購入した。 GQlbは Alexi s Biochemicals社から購入した。 CMP - [14C] - NeuAc (12. 0 GBq/mmol)は Amersham Pharmacia Biotech社力 ら購人した。 シァリダーゼ (NANase II， III)は Glyko Inc社から購入した。 V-glycanase (Glycopeptidase F)は宝酒 造から購入した。 [ a - 32P] dCTPは NEN社から購入した。 ヒ h Multiple tissue cDNA panelは Clontech社から購入した。 GMlb， およびその positional analogである GSし— 68, 2, 3一 sia丄 ylparagloboside (2, 3一 SPG) , 2, D-sialylparagloboside (2, 6- SPG)は木曽真教授 (岐阜大学農学部) から、 NeuAc α 2, 3Gal, NeuAc α 2, 6Gal は石田秀樹博士 （野口研究所） から寄贈されたものを使用した。 抗 GD3モノクロ ーナル抗体 KM641は協和発酵、 設楽研也、 花井陳雄両博士から寄贈されたものを 使用した。また抗 NeuAc a 2, 8NeuAc a 2, 3Gal抗体 S2-566は生化学工業より購入し 7こ。 Peroxidase-conjugated Aff iPure goat anti-mouse IgG+IgM (H+L は Jackson ェ議 no Research社力 ら購 vした。 BSM, ο; 1 - acid glycoprotein, ovomucoidの脱ン アル化 （ァシァ口） 糖タンパク質は、 これらを 0. 02N HC1中 80度、 1時間で処理 することにより調製した。 The reagents and samples used in the specific examples of the present invention are as follows. Fetuin, asialofetuin, bovine submaxi llary mucin (BSM), a 1-acid glycoprotein, ovomucoid, lactosyl ceramide (LacCer), GM3, GMla, GDI a, GDlb, iiTlb, CMP-NeuAc, 6'-sialyllactose, 3'-sial -vV-acetyllactosamine and Triton CF-54 were purchased from Sigma. 3'-sialyllactose, 6'-sialyl-jV-acetyllactosamine was purchased from Calbiochem. -Acetylneuraminic acid (NeuAc), G4, Gal, N-Acetylgalactosamine (GalNAc) was purchased from Wako Pure Chemical Industries. GD3 was purchased from Snow Brand Milk Products. GQlb was purchased from Alexis Biochemicals. CMP-[ 14 C]-NeuAc (12.0 GBq / mmol) was purchased from Amersham Pharmacia Biotech. Sialidase (NANase II, III) was purchased from Glyko Inc. V-glycanase (Glycopeptidase F) was purchased from Takara Shuzo. [a- 32 P] dCTP was purchased from NEN. H Multiple tissue cDNA panel was purchased from Clontech. GMlb and its positional analog, GS-68,2,3-I sia 丄 ylparagloboside (2,3-I SPG) and 2, D-sialylparagloboside (2,6-SPG) were obtained from Professor Kiso Makoto (Faculty of Agriculture, Gifu University) For NeuAc α 2, 3Gal and NeuAc α 2, 6Gal, those donated by Dr. Hideki Ishida (Noguchi Laboratory) were used. The anti-GD3 monoclonal antibody KM641 used was donated by Drs. Kyowa Hakko, Kenya Shitara and Chen Yu Hanai. Anti-NeuAc a 2, 8 NeuAc a 2, 3Gal antibody S2-566 was purchased from Seikagaku Corporation. Peroxidase-conjugated Aff iPure goat anti-mouse IgG + IgM (H + L purchased from Jackson Research No Research, Inc. BSM, ο; 1-acid glycoprotein, ovomucoid deamination (Asia mouth) glycoprotein Were prepared by treating them in 0.02N HC1 at 80 degrees for 1 hour.
マウスシアル酸転移酵素 ST8Sia Vのァミノ酸配列を用いて、 これと相同性を示 す新規シアル酸転移酵素をコードしているクローンを National Center for Biotechnology Information < expressed sequence tag (dbESi)のテータへ1 ~ス で検索したところ、 GenBank™ accession Nos. BE633149, BE686184, BF730564の 各クローンが得られた。 これらの塩基配列情報をもとに 2 種類の合成 DNA, 5' -CTTTTCTGGAGAACTAAAGG-3' (図 1 Aの塩基番号 1001- 1020に相当）（配列番号 9 ) , 5' -AATTGCAGTTTGAGGATTCC-3' (図 1 Aの塩基番号 1232-1251の相補鎖に相当） （配 列番号 1 0 ) を作製し、 Israelの方法に従い（Israel, D. I. (1993) Nucleic Acids Res. 21, 2627-2631) , ポリメラーゼ連鎖反応法 (PCR)を利用してマウス脳および 心臓の各 cDNAライブラリーをスクリーニングしたところ、新規シアル酸転移酵素 の一部をコードしているクローンがそれぞれの cDNAライブラリ一から 1個ずつ得 られた。 全長ク ロ ーンを得る ため、 さ ら に 2 種類の合成 DNA, 5' -TGGCTCAGGATGAGATCGGG-3' (図 1 Aの塩基番号 68-87に相当）（配列番号 1 1 ) , 5' -TACTAGCGCTCCCTGTGATTGG-3' (図 1 Aの塩基番号 725-746の相補鎖に相当） （配 列番号 1 2 ) を作製し、 マウス腎臓由来 cDNAを铸型として PCR法により両合成 DNA間部分の DNAを増幅した。 この増幅断片とマウス脳 cDNAライブラリーから得 られたクローンを連結することにより、 全長クローンを得た。 この cDNA は 398 アミノ酸からなる予測分子量 45, 399の II型膜タンパク質をコードする単一の翻 訳領域を有していた。 またそのアミノ酸配列にはシアル酸転移酵素に保存されて いるシァリルモチーフが存在していた。 本タンパク質は既知マウスシアル酸転移 酵素の中では ST8Sia I, Vとそれぞれァミノ酸配列レベルで 42. 0%, 38. 3%の相同 性を示した（図 2 A)。 なお以下に示すようにこのタンパク質は α 2, 8-シアル酸転 移酵素活性を有していたことから、 これを本発明の i^glycan α2, 8_シアル酸転移 酵素 ST8Sia VIと命名した。 Using Amino acid sequence of mouse sialyltransferase ST8Sia V, which homology to clone the encoding shown to novel sialyltransferase into stator of National Center for Biotechnology Information <expressed sequence tag (dbESi) 1 As a result, clones of GenBank ™ accession Nos. BE633149, BE686184, and BF730564 were obtained. Based on these base sequence information, two types of synthetic DNA, 5'-CTTTTCTGGAGAACTAAAGG-3 '(corresponding to base numbers 1001-1020 in FIG. 1A) (SEQ ID NO: 9), 5'-AATTGCAGTTTGAGGATTCC-3' (corresponding to the complementary strand of base numbers 1232-1251 in FIG. 1A) ( SEQ ID NO: 10) was prepared, and mouse brain and heart cDNAs were synthesized by polymerase chain reaction (PCR) according to the method of Israel (Israel, DI (1993) Nucleic Acids Res. 21, 2627-2631). Screening the libraries yielded one clone from each cDNA library, encoding a portion of the novel sialyltransferase. To obtain a full-length clone, two additional types of synthetic DNA, 5'-TGGCTCAGGATGAGATCGGG-3 '(corresponding to base numbers 68-87 in FIG. 1A) (SEQ ID NO: 11), 5'-TACTAGCGCTCCCTGTGATTGG- 3 ′ (corresponding to the complementary strand of base numbers 725-746 in FIG. 1A) (SEQ ID NO: 12) was prepared, and the DNA between the synthetic DNAs was amplified by PCR using mouse kidney-derived cDNA as type III. . This amplified fragment was ligated to a clone obtained from a mouse brain cDNA library to obtain a full-length clone. This cDNA had a single translated region encoding a type II membrane protein of 398 amino acids with a predicted molecular weight of 45,399. The amino acid sequence contained a sialyl motif conserved in sialyltransferase. This protein showed 42.0% and 38.3% homology with ST8Sia I and V among known mouse sialyltransferases at the amino acid sequence level, respectively (Fig. 2A). Since this protein had α2,8-sialyltransferase activity as shown below, it was named i ^ glycan α2,8_sialyltransferase ST8Sia VI of the present invention.
一方、他の哺乳動物においてもこれと同様の酵素が存在するのかを調べるため、 マウス ST8Sia VIの配列情報を利用して、 上記と同様にデータベースを検索した ところ、 ヒ トゃラットにも同様の酵素が存在することが確認できた。 図 1 Bにヒ トの ST8Sia VIの配列情報を示す。 マウスとヒ トの ST8Sia VIではァミノ酸配列 レベルで 82. 4%の相同性を示した（図 2 B )。  On the other hand, a database was searched in the same manner as above using the sequence information of mouse ST8Sia VI to determine whether the same enzyme exists in other mammals. It was confirmed that the enzyme was present. Figure 1B shows the sequence information of human ST8Sia VI. ST8Sia VI of mouse and human showed 82.4% homology at the amino acid sequence level (FIG. 2B).
つぎに ST8Sia VIの酵素学的諸性質を調べるため、 分泌型タンパク質の製造を 行った。 まずマウス ST8Sia VIについて、 それぞれ ¾oIサイ トを含む 2種類の合 成 DNA, 5' - TGCTCTCGAGCCCAGCCGACGCGCCTGCCC- 3' (図 1 Aの塩基番号 141-170に 相当） （配列番号 1 3 ) , 5' -TATTCTCGAGCTAAGAAACGTTAAGCCGTT-3' (図 1 Aの塩基 番号 1263-1293の相補鎖に相当） （配列番号 1 4 ) を用い、 クローニングした全 長 cDNAを铸型として PCR法により、マウス ST8Sia VIの活性ドメインをコードす る DNA断片を増幅した。これを J ?り Iで切断後、哺乳動物発現べクタ一 pcDSAの ¾oI サイ 卜に挿入した。 この発現ベクターを pcDSA - mST8Sia VIと命名した。 Next, secretory proteins were produced to examine the enzymatic properties of ST8Sia VI. First, for mouse ST8Sia VI, two types of synthetic DNA, each containing an oI site, 5'-TGCTCTCGAGCCCAGCCGACGCGCCTGCCC-3 '(see base numbers 141-170 in Figure 1A) (SEQ ID NO: 13), 5'-TATTCTCGAGCTAAGAAACGTTAAGCCGTT-3 '(corresponding to the complementary strand of base number 1263-1293 in FIG. 1A) (SEQ ID NO: 14) A DNA fragment encoding the active domain of mouse ST8Sia VI was amplified by PCR. This was digested with J II and inserted into the ¾oI site of the mammalian expression vector pcDSA. This expression vector was named pcDSA-mST8Sia VI.
またヒ ト ST8Sia VIについては、まず Human Tumor Multiple Tissue cDNA Panels (Clontech)の Colon adenocarcinoma CX-1由来 cDNAを铸型として、 2種類の合成 DNA, 5' - CAATTGACATATCTGAATGAGMGTCGCTC- 3' (図 1 Bの塩基番号 293- 315 に相 当） (配列番号 1 5 ) , 5' -TACTAACATCTCCTGTGGTTGG-3' (図 1 Bの塩基番号 740- 761 の相補鎖に相当） （配列番号 1 6 ) を用いて PCR法により増幅した DNA断片、 お よび 2種類の合成 DNA, 5' -CCAGTGTCCCAGCCTTTTGT-3 ' (図 1 Bの塩基番号 608-627 に相当） （配列番号 1 7 ) , 5' -TGAGTGGGGAAGCTTTGGTC-3' (図 1 Bの塩基番号 1407- 1426の相補鎖に相当） （配列番号 1 8 ) を用いて PCR法により増幅した DNA 断片を、 両増幅 DNA 断片が共通に有する oRI サイ トを利用して連結し、 ヒ ト ST8Sia VIの活性ドメインをコードする DNA断片を得た。 これをクローニングべ クタ一 pBluescript I I SK (+)の oRVサイ トに挿入した後、 と ¾oIで切り 出し、 この切り出し断片を pcDSAの £coRI- ¾Iサイ トに挿入したものを、発現べ クタ一 pcDSA - hST8Sia VIと命名した。  For human ST8Sia VI, first, cDNA derived from Colon adenocarcinoma CX-1 of Human Tumor Multiple Tissue cDNA Panels (Clontech) was used as type II, and two types of synthetic DNA, 5′-CAATTGACATATCTGAATGAGMGTCGCTC-3 ′ (base in FIG. No. 293-315) (SEQ ID NO: 15), 5'-TACTAACATCTCCTGTGGTTGG-3 '(corresponding to the complementary strand of base Nos. 740-761 in FIG. 1B) (SEQ ID NO: 16) Amplified DNA fragment and two types of synthetic DNA, 5'-CCAGTGTCCCAGCCTTTTGT-3 '(corresponding to base numbers 608-627 in FIG. 1B) (SEQ ID NO: 17), 5'-TGAGTGGGGAAGCTTTGGTC-3' (FIG. 1 The DNA fragments amplified by the PCR method using (SEQ ID NO: 18) were ligated using the oRI site shared by both amplified DNA fragments. A DNA fragment encoding the active domain of ST8Sia VI was obtained. After inserting this into the oRV site of the cloning vector pBluescript II SK (+) and excising with ¾oI, the excised fragment inserted into the £ coRI-¾I site of pcDSA was inserted into the expression vector pcDSA. -Named hST8Sia VI.
PcDSA-mST8Sia VIおよび pcDSA- hST8Sia VIは、 それぞれマウス免疫グロブリ ン IgMのシグナノレぺプチドと Staphylococcus aureus protein A, およびマウス またはヒ ト ST8Sia VIの活性ドメイン（マウス ST8Sia VIではァミノ酸番号 26-398、 ヒト ST8Sia VIではアミノ酸番号 68-398)からなる分泌型融合タンパク質をコー ドする。 P cDSA-mST8Sia VI and pcDSA- hST8Sia VI is Shigunanore peptides of each mouse immunoglobulin down IgM and Staphylococcus aureus protein A, and a mouse or human ST8Sia VI of the active domain (mouse ST8Sia VI in Amino acid numbers 26-398, Human ST8Sia VI encodes a secretory fusion protein consisting of amino acids 68-398).
各発現ベクターとリボフェク トァミン（Invitrogen)を用いて COS- 7細胞でその 一過性発現を行った（Kojima, N. et al. (1995) FEBS Lett. 360, 1-4)。 ここで それぞれの発現ベクターを導入した細胞から細胞外に分泌された本発明のタンパ ク質を PA- mST8Sia VI (マウス）および PA- hST8Sia VI (ヒ ト）と命名した。 PA-mST8Sia VI、 PA_hST8Sia VIは IgG-Sepharose (Amersham Pharmacia Biotech 社）に吸着させて培地より回収した。シアル酸転移酵素活性は Leeらの方法に準じ て以下のよ う に行った（Lee, Y. -C. et al. (1999) J. Biol. Chem. 274, 11958 - 11967)。 50 raM MESバッファー（pH 6.0) , 1 raM gCl2, 1 mM CaCl2, 0.5% Triton CF-54, 100 μΜ CMP- [14C]_NeuAc, 糖鎖（糖脂質の場合は 0.5 mg/ral, 糖タンパク質、 オリゴ糖は 1 mg/mlになるように添加）、 および PA - mST8SiaVIまたは PA- hST8Sia VI懸濁液を含む反応液（10 μ 1)を 37度で 3-20時間ィンキュベートし、 その後、 糖脂質については C- 18カラム（Sep- Pak Vac 100 mg; Waters社）を用いて精製し たものを試料として、 オリゴ糖、 糖タンパク質については反応産物をそのまま試 料として解析を行った。 オリゴ糖、 糖脂質はシリカゲル 60HPTLCプレート（Merck 社）にスポッ トし、エタノール：ピリジン： n -ブタノール：水：酢酸 =100:10:10:30:3 の展開溶媒 （オリゴ糖用）、 または 1-プロパノール：アンモニア水：水 =6:1:2.5 の展開溶媒 （オリ ゴ糖用） 、 またはクロ口ホルム ： メ タノール ： 0.02% CaCl2=55:45:10の展開溶媒 （糖脂質用） で展開した。 糖タンパク質の場合は SDS- ポリアクリルアミ ドゲル電気泳動によって解析を行った。 これらの放射活性を BAS2000ラジオイメージアナライザー （フジフィルム） で可視化し、 定量した。 表 1に PA- mST8Sia VI、 PA-hST8Sia VIの基質特異性を示す。 Using each expression vector and ribofectamine (Invitrogen), the cells were transiently expressed in COS-7 cells (Kojima, N. et al. (1995) FEBS Lett. 360, 1-4). Here, the proteins of the present invention secreted extracellularly from the cells into which the respective expression vectors were introduced were named PA-mST8Sia VI (mouse) and PA-hST8Sia VI (human). PA-mST8Sia VI and PA_hST8Sia VI were adsorbed on IgG-Sepharose (Amersham Pharmacia Biotech) and recovered from the medium. Sialyltransferase activity was performed as follows according to the method of Lee et al. (Lee, Y.-C. et al. (1999) J. Biol. Chem. 274, 11958-11967). 50 raM MES buffer (pH 6.0), 1 raM gCl 2 , 1 mM CaCl 2 , 0.5% Triton CF-54, 100 μΜ CMP- [ 14 C] _NeuAc, sugar chain (0.5 mg / ral for glycolipid, sugar Incubate the reaction solution (10 μl) containing the protein and oligosaccharide at a concentration of 1 mg / ml) and PA-mST8SiaVI or PA-hST8SiaVI suspension at 37 ° C for 3-20 hours. Glycolipids were analyzed using a sample purified using a C-18 column (Sep-Pak Vac 100 mg; Waters), and oligosaccharides and glycoproteins were analyzed using the reaction product as a sample. Oligosaccharides and glycolipids are spotted on silica gel 60HP TLC plates (Merck), and developing solvents (for oligosaccharides) of ethanol: pyridine: n-butanol: water: acetic acid = 100: 10: 10: 30: 3 or 1 -Propanol: Ammonia water: Water = 6: 1: 2.5 developing solvent (for oligosaccharide) or Cloh form: Methanol: 0.02% CaCl 2 = 55: 45: 10 developing solvent (for glycolipid) Expanded. For glycoproteins, analysis was performed by SDS-polyacrylamide gel electrophoresis. The radioactivity was visualized and quantified using a BAS2000 radio image analyzer (Fujifilm). Table 1 shows the substrate specificity of PA-mST8Sia VI and PA-hST8Sia VI.
表 l srresiaviの受容体基質特異性 Table l Receptor substrate specificity of srresiavi
PA-mSrr8SiaVIおよび PA- hS 8SiaVIを用いて様々な受容体基質に対する特異性を検討した。 各基質の濃度は、糖脂質の場合は 0.5 mg/mlに、 糖夕ンパク質、 単糖、 オリゴ糖の場合は 1 mg/mlになるようにした。 相対活性は Fetuinの取り込み値 PA - mST8Sia VIは 2.06 pmol/h/(ml酵 素液 PA- S 8SiaVIは 0.204 pmol/h/(ml酵素液))を 100として計算した。 Rは W型糖鎖の残りの糖鎖部分を意味する。 ND：測定せず。  PA-mSrr8SiaVI and PA-hS8SiaVI were used to examine the specificity for various receptor substrates. The concentration of each substrate was 0.5 mg / ml for glycolipids and 1 mg / ml for glycoproteins, monosaccharides and oligosaccharides. The relative activity was calculated based on the Fetuin uptake value PA-mST8Sia VI of 2.06 pmol / h / (ml enzyme solution PA-S8SiaVI of 0.204 pmol / h / (ml enzyme solution)). R means the remaining sugar chain part of the W-type sugar chain. ND: Not measured.
Acceptor Representative structures oi carbohydrates . Relative rale (%)  Acceptor Representative structures oi carbohydrates .Relative rale (%)
Mouse Human ST8Sia VI ST8Sia VI Mouse Human ST8Sia VI ST8Sia VI
Glycoproteins Glycoproteins
Fetuin NeuAca2,3Gai 1 ,3GalNAc-0-Ser Thr 100 100  Fetuin NeuAca2,3Gai 1, 3GalNAc-0-Ser Thr 100 100
NeuAca2,3Gai i,3(NeuAca2,6)GalNAc-0-Ser Thr  NeuAca2,3Gai i, 3 (NeuAca2,6) GalNAc-0-Ser Thr
NeuAca2,6(3)Gai i,4GlcNAc-R  NeuAca2,6 (3) Gai i, 4GlcNAc-R
Asialofetuin 0 0 al-Acid glycoprotein NeuAca2,6(3)Gai ! ,4GlcNAc-R 0 0 Asialofetuin 0 0 al-Acid glycoprotein NeuAca2,6 (3) Gai!, 4GlcNAc-R 0 0
Asialo- al-Acid glycoprotein 0 0Asialo- al-Acid glycoprotein 0 0
BSM NeuAca2,6GalNAc-0-Ser/Thr 375 24.2 BSM NeuAca2,6GalNAc-0-Ser / Thr 375 24.2
GlcNAcP 1 ,3(NeuAca2,6)GalNAc-0-Ser hr  GlcNAcP 1,3 (NeuAca2,6) GalNAc-0-Ser hr
Asialo-BSM 0 0 Asialo-BSM 0 0
Ovomucoid NeuAca2,3Gaip 1 ,4GlcNAc-R 6.2 12.3Ovomucoid NeuAca2,3Gaip 1,4GlcNAc-R 6.2 12.3
Asialoovomucoid 0 0Asialoovomucoid 0 0
Glycolipids Glycolipids
Lactosylceramide Gaipi,4Glc l-Cer 0 ND Lactosylceramide Gaipi, 4Glc l-Cer 0 ND
G 4 NeuAca2,3Gai i-Cer 1.0 NDG 4 NeuAca2,3Gai i-Cer 1.0 ND
GM3 Neu Aca2.3Gaip 1 ,4Glcp 1 -Cer 13.0 1.6GM3 Neu Aca2.3Gaip 1, 4Glcp 1 -Cer 13.0 1.6
GMla Galpl ,3GalN Α。β 1 ,4(NeuAca2,3)Gai i ,4Glc& 1 -Cer 0 NDGMla Galpl, 3GalN Α. β 1, 4 (NeuAca2,3) Gai i, 4Glc & 1 -Cer 0 ND
GDla 6.0 1.8GDla 6.0 1.8
GD3 0 0GD3 0 0
GDlb 0 NDGDlb 0 ND
GTlb 1.1 2.2GTlb 1.1 2.2
GQlb 0 0GQlb 0 0
GMlb 1.0 NDGMlb 1.0 ND
GSC-68 2.6 NDGSC-68 2.6 ND
2,3-SPG 3.5 ND2,3-SPG 3.5 ND
2,6-SPG
Figure imgf000032_0001
0.98 ND 2,6-SPG
Figure imgf000032_0001
0.98 ND
Monosaccharides and oligosaccharides Monosaccharides and oligosaccharides
3'-SialyUactose NeuAca2.3Galpl.4Glc 629 69.9 6'-Sialyllactose NeuAca2.6Gal 1 ,4Glc 91.5 10.7 3'-SialyUactose NeuAca2.3Galpl.4Glc 629 69.9 6'-Sialyllactose NeuAca2.6Gal 1, 4Glc 91.5 10.7
S^Sialyl-TV-acetyllactosamine NeuAca2f3Galp 1 ,4GlcNAc 411 ND 6，- Sialyl- /-acetyllaclosamine NeuAca2,6Gal 1.4GlcNAc 88.7 NDS ^ Sialyl-TV-acetyllactosamine NeuAca2 f 3Galp 1, 4GlcNAc 411 ND 6, -Sialyl-/-acetyllaclosamine NeuAca2,6Gal 1.4GlcNAc 88.7 ND
3'-Sialylgalactose NeuAca2,3Gal 13.9 ND 6*-Sialylgalactose NeuAca2,6Gal 2.0 ND V-Acetylneuraminic acid NeuAc 0 ND Galactose Gal 0 ND3'-Sialylgalactose NeuAca2,3Gal 13.9 ND 6 * -Sialylgalactose NeuAca2,6Gal 2.0 ND V-Acetylneuraminic acid NeuAc 0 ND Galactose Gal 0 ND
//-Acetylgalactosamine GalNAc 0 ND //-Acetylgalactosamine GalNAc 0 ND
t t
PA-mST8Sia VIは GM4, GM3, GDla, GTlb, GMlb, GSC- 68, 2, 3- SPG， 2， 6- SPGな ど、非還元末端に NeUACa2, 3 (6) Gal-という構造をもつ糖脂質に対して活性を示し た。 このうち GM3を基質とした場合、 その反応産物は α 2, 3-， α 2， 6-結合で結合 しているシアル酸を特異的に切断するシァリダーゼ（NANase II)では導入シアル 酸が切断されなかったが、 α 2, 3-, a 2, 6-, ct 2, 8_， a 2， 9-結合で結合している シアル酸を特異的に切断するシァリダーゼ（NANase III)では、 導入シアル酸が切 断された（図 3 A)。 またこの反応産物は抗 GD3モノクローナル抗体 KM641を用い た TLC免疫染色（Saito, M. et al. (2000) Biochim. Biophys. Acta 1523, 230 - 235) によって α2, 8 結合を介してシアル酸が導入された GD3 であることが確認された ことから（図 3 B )、 PA-mST8Sia VIはシアル酸を α2, 8-の結合様式で転移すること が明らかになった。 PA-mST8Sia VI is called Ne U A Ca 2,3 (6) Gal- at the non-reducing end, such as GM4, GM3, GDla, GTlb, GMlb, GSC-68, 2, 3-SPG, 2, 6-SPG It showed activity on glycolipids having a structure. When GM3 is used as a substrate, the reaction product is cleaved by sialidase (NANase II), which specifically cleaves sialic acid bound by α2,3-, α2,6-linkage. However, sialidase (NANase III), which specifically cleaves sialic acid bound by α 2,3-, a 2,6-, ct 2,8_, a 2,9-bond, Was cut off (Fig. 3A). Sialic acid was introduced into this reaction product via α2,8 linkage by TLC immunostaining using the anti-GD3 monoclonal antibody KM641 (Saito, M. et al. (2000) Biochim. Biophys. Acta 1523, 230-235). The confirmed GD3 was confirmed (Fig. 3B), indicating that PA-mST8Sia VI transfers sialic acid in an α2,8 binding mode.
一方、 糖タンパク質を基質とした場合（表 1)、 PA-mST8Sia VIは 型糖鎖のみを 含有する BSM に対して最も高い活性を示した。 型糖鎖、 型糖鎖を含有する Fetuin、 型糖鎖のみを含有する Ovomucoidに対しても活性を示したが、 Ovomucoid に対する活性は 型糖鎖を含むタンパク質に比べると低かった。なお、 PA- mST8Sia VIはァシァ口糖タンパク質に対しては全く活性を示さなかった。 また単糖および オリゴ糖を基質とした実験により（表 1)、 PA - mST8Sia VIが基質として認識する最 小糖鎖単位は NeuAc o; 2, 3 (6) Galであることが明らかになった。  On the other hand, when glycoprotein was used as a substrate (Table 1), PA-mST8Sia VI showed the highest activity against BSM containing only type-glycans. It also showed activity against type sugar chains, Fetuin containing type sugar chains, and Ovomucoid containing only type sugar chains, but the activity against Ovomucoid was lower than that of proteins containing type sugar chains. In addition, PA-mST8Sia VI did not show any activity against asia oral glycoprotein. In addition, experiments using monosaccharides and oligosaccharides as substrates (Table 1) revealed that the minimum sugar chain unit recognized by PA-mST8Sia VI as a substrate was NeuAco; 2, 3 (6) Gal. .
Fetuinを基質としたとき、 PA- mST8Sia VIによってあらたに導入されたシアル 酸の大部分は 型糖鎖に取り込まれていることが - glycanase処理によって明ら かになつた（図 4 )。 すなわち PA-mST8Sia VIを用いて Fetuinを ["C] -NeuAcでシ アル化し、 これを 型糖鎖をぺプチド部分から遊離する -glycanaseで処理する と、 大部分 (82. 7%)の放射能活性はこの Fetuinに保持されたままであった。 この ことは PA_mST8Sia VIによって導入されたシアル酸の大部分は 型糖鎖に取り込 まれたことを示す。 一方、 型糖鎖を受容体基質とするマウス STSSia IIIを用い て同様の実験を行ったところ、 放射活性は完全に消失した。  When Fetuin was used as a substrate, most of the sialic acid newly introduced by PA-mST8Sia VI was incorporated into the type sugar chain-it was clarified by glycanase treatment (Fig. 4). In other words, Fetuin is sialylated with ["C] -NeuAc using PA-mST8Sia VI and treated with -glycanase, which releases the type sugar chain from the peptide moiety. Activity was retained by this Fetuin, indicating that most of the sialic acid introduced by PA_mST8Sia VI was incorporated into the type sugar chain, while the type sugar chain was recognized as the receptor substrate. When a similar experiment was performed using mouse STSSia III, the radioactivity completely disappeared.
さらに PA- mST8Sia VIの基質特異性および選択性を明らかにするため、 BSM と GM3に対する Km値、 V腿値を求めた。 BSMに対しては Km値 =0. 03 mM, Vwax値 =23. 8 pmol/h/ (ml酵素液）で、 ½ra / A¾値は 793であった。 一方、 GM3に対しては^¾値 =0. 5 mM, Vwax値 =0. 67pmol/h/ (ml酵素液）で、 Vmax/Km値は 1. 34であつた。 以上 の結果は、 PA-mST8Sia VIにとつて 型糖鎖が糖脂質や 型糖鎖よりはるかに好 ましい基質であることを示している。 To further clarify the substrate specificity and selectivity of PA-mST8Sia VI, The Km value and V thigh value for GM3 were determined. For BSM, Km value = 0.03 mM, Vwax value = 23.8 pmol / h / (ml enzyme solution), and ½ra / A¾ value was 793. On the other hand, for GM3, ^ ¾ value = 0.5 mM, Vwax value = 0.67 pmol / h / (ml enzyme solution), and Vmax / Km value was 1.34. These results indicate that PA-mST8Sia VI is a much better substrate for type sugar chains than glycolipids and type sugar chains.
上記の酵素学的諸性質については、活性値に多少の差はあるものの PA- hST8Sia VIについても当てはまることから（表 1、 図 3 A、 図 4 )、各種動物由来の ST8Sia VI が従来の α 2， 8 -シアル酸転移酵素とは異なる基質特異性を有することが示さ れたといえる。  Regarding the above enzymatic properties, although there are some differences in the activity values, they also apply to PA-hST8Sia VI (Table 1, Figure 3A, Figure 4). It can be said that it has been shown to have a different substrate specificity from 2,8-sialyltransferase.
またマウス ST8Sia VIについては、 その全長クローンの細胞内における酵素活 性についても調べた（図 5 )。マウス ST8Sia VIの全長をコードする領域を含む 1. 4 kbの oil-^oal断片を、 発現ベクター pRc/CMVの o l- サイ 卜に挿入したも のを pRc/CMV- ST8Sia VIと命名し、 これをリポフエクトアミンを用いて COS- 7細 胞に導入した。 この細胞よりガングリオシドを抽出し、 NeuAc a 2, 8NeuAc α 2, 3Gal 構造を認識するモノクローナル抗体 S2-566を用いて TLC免疫染色を行ったところ (図 5 A)、 pRc/CMV- ST8Sia VI導入細胞において有意に NeuAc α 2, 8NeuAc α 2, 3Gal 構造を有するガングリオシド量が増加していたことが明らかになった。 また細胞 内の糖タンパク質についても、 pRc/CMV- ST8Sia VI導入細胞では 型糖鎖上に新 たに NeuAco 2, 8NeuAca2，3Gal構造が形成されていた（図 5 B )。 以上の結果は、 マ ウス ST8Sia VIが生体内において α 2， 8 -シアル酸転移酵素として機能しているこ とを示している。  For mouse ST8Sia VI, the enzyme activity in the cells of the full-length clone was also examined (Fig. 5). A 1.4 kb oil- ^ oal fragment containing the entire region encoding mouse ST8Sia VI was inserted into the o-site of the expression vector pRc / CMV and named pRc / CMV-ST8Sia VI. This was introduced into COS-7 cells using lipofectamine. Gangliosides were extracted from these cells and subjected to TLC immunostaining using the monoclonal antibody S2-566 recognizing the NeuAc a 2,8 NeuAc α2,3Gal structure (Fig.5A) .The cells transfected with pRc / CMV-ST8Sia VI It was found that the amount of gangliosides having a NeuAc α2,8NeuAc α2,3Gal structure was significantly increased. Regarding the intracellular glycoprotein, in the cells transfected with pRc / CMV-ST8Sia VI, new NeuAco2,8NeuAca2,3Gal structures were formed on the type sugar chains (Fig. 5B). The above results indicate that mouse ST8Sia VI functions as α2,8-sialyltransferase in vivo.
なお、マゥス ST8Sia VIは腎臓、心臓、脾臓などで主に発現してレ、るが（図 6 A)、 ヒ ト ST8Sia VIは胎盤や胎児の各種臓器、 および各種腫瘍細胞などにおいて主に 発現している（図 6 B )。 実施例 2 : j3—ガラク トシド α 2 , 6 —シアル酸転移酵素  Although mouse ST8Sia VI is mainly expressed in kidney, heart, spleen, etc. (Fig. 6A), human ST8Sia VI is mainly expressed in various organs of the placenta and fetus, and various tumor cells. (Fig. 6B). Example 2: j3-galactoside α2,6-sialyltransferase
本発明の具体例に用いた試薬、 試料類は以下の通りである。 Fetuin, asialofetuin, bovine submaxillary mucin (BSM) , o l - acid glycoprotein, ovomucoid, lactosyl ceramide (LacCer) , GA1, GM3, GMla, Gai i, 3GalNAc, Galpl, 3GlcNAc, Galpl, 4GlcNAc, Triton CF- 54， β -ガラク トシダーゼ (牛精巣由 来） は Sigma社から購入した。 Paragloboside, ラタ トースは和光純薬から購入し た。 CMP- [14C] - NeuAc (12. 0 GBq/mmol)は Amersham Pharmacia Biotech社力 ら購 入した。 Lacto-^-tetraose, Lacto-^-neotetraose, シァリダーゼ (NANase I, II) は Glyko Inc社から購入した。 [a- 32P] dCTPは NEN社から購入した。 ヒ トおよび マウス Multiple tissue cDNA panelは Clontech社力 ら購入した。 BSM, al-acid glycoprotein, ovomucoid の脱シアル化 （ァシァ口） 糖タンパク質は、 これらを 0. 02N HC1中 80度、 1時間で処理することにより調製した。 The reagents and samples used in the specific examples of the present invention are as follows. Fetuin, MN US asialofetuin, bovine submaxillary mucin (BSM), ol-acid glycoprotein, ovomucoid, lactosyl ceramide (LacCer), GA1, GM3, GMla, Gai i, 3GalNAc, Galpl, 3GlcNAc, Galpl, 4GlcNAc, Triton CF-54, β-lacto (Bovine testes) was purchased from Sigma. Paragloboside and rata tose were purchased from Wako Pure Chemical. CMP- [ 14 C] -NeuAc (12.0 GBq / mmol) was purchased from Amersham Pharmacia Biotech. Lacto-^-tetraose, Lacto-^-neotetraose, and sialidase (NANase I, II) were purchased from Glyko Inc. [a- 32 P] dCTP was purchased from NEN. Human and mouse Multiple tissue cDNA panels were purchased from Clontech. BSA, al-acid glycoprotein, and ovomucoid desialylated glycoside glycoproteins were prepared by treating them in 0.02N HC1 at 80 ° C for 1 hour.
ヒ トシアル酸転移酵素 ST6Gal Iのアミノ酸配列を用いて、 これと相同性を示す 新規シアル酸転移酵素をコードしているクローンを National Center for Biotechnology 丄 niormationの expressed sequence tag (dbJiST)のァータベース で検索したところ、 GenBank™ accession Nos. BE613250, BE612797, BF038052の 各 ESTクローンが得られた。 これらについては I. M. A. G. E. Consortiumより 該当クローンを入手した。 またそれらの塩基配列情報を利用して、 さらに dbEST とヒ トクノムの High throughput genomic sequenceのデータべ一スを恢索しにと ころ、 関連 EST クローンとゲノム遺伝子の塩基配列情報が得られた （Accession Nos. H94068, AA514734, BF839115, AA210926, AA385852, H94143, BF351512 (以 上 ESTクローン）， AC016994 (ゲノム配列） ）。 以上の塩基配列情報をもとにポ リメラーゼ連鎖反応法 （PCR)用のプライマーを作製し、ヒ ト大腸由来 cDNAを铸型 として PCRを行い、 ここで得られた増幅断片と入手 ESTクローン由来の DNA断片 を連結することによって翻訳領域全長を含むクローンを得た（図 7 A)。この cDNA は 529アミノ酸からなる予測分子量 60, 157の II型膜タンパク質をコードする単 —の翻訳領域を有していた。 なお膜貫通ドメィンは疎水性分布図によりアミノ酸 番号 12 - 30の領域に存在することが予測された （図 7 B ) 。 本タンパク質のアミ ノ酸配列にはシアル酸転移酵素に保存されているシァリルモチーフが存在してい た。 また本タンパク質は既知ヒ トシアル酸転移酵素の中では ST6Gal Iとアミノ酸 レベルで最も高い相同性（48. 9%) を示したが（図 9 A)、他のファミリーのシアル 酸転移酵素とは 21-36%程度の相同性を示したに過ぎなかった。なお以下に示すよ うにこのタンパク質は /3 -ガラク トシドひ 2, 6-シアル酸転移酵素活性を有してい たことから、 これを本発明の /3 -ガラク トシド α 2, 6-シアル酸転移酵素 ST6Gal II と命名した。 またヒ ト ST6Gal IIには、 splicing variantと考えられるシァリル モチーフ Sの途中から配列が異なる short formのクローンも存在していた（図 7 A)。 Using the amino acid sequence of human sialyltransferase ST6Gal I, a clone encoding a novel sialyltransferase showing homology to this was searched for in the database of the expressed sequence tag (dbJiST) of the National Center for Biotechnology 丄 niormation. However, each EST clone of GenBank ™ accession Nos. BE613250, BE612797, BF038052 was obtained. For these, the corresponding clones were obtained from the IMAGE Consortium. In addition, using these base sequence information, the base sequence information of related EST clones and genomic genes was obtained in order to further retrieve the database of dbEST and the high throughput genomic sequence of cytochrome. Nos. H94068, AA514734, BF839115, AA210926, AA385852, H94143, BF351512 (above EST clone), AC016994 (genomic sequence)). Based on the above nucleotide sequence information, primers for polymerase chain reaction (PCR) were prepared, and PCR was performed using human colon-derived cDNA as type II. The amplified fragment obtained here and the derived EST clone-derived clone were used. By ligating the DNA fragments, a clone containing the entire translation region was obtained (FIG. 7A). This cDNA had a single translation region encoding a type II membrane protein consisting of 529 amino acids and having a predicted molecular weight of 60,157. The transmembrane domain was predicted to be present in the region of amino acids 12 to 30 according to the hydrophobic distribution diagram (FIG. 7B). The amino acid sequence of this protein contains a sialyl motif conserved by sialyltransferase. Was. Although this protein showed the highest homology (48.9%) at the amino acid level with ST6Gal I among known human sialyltransferases (Fig. 9A), it did not differ from other families of sialyltransferases. It showed only -36% homology. As shown below, this protein had a / 3-galactosidyl 2,6-sialyltransferase activity. The enzyme was named ST6Gal II. In human ST6Gal II, there was also a short form clone with a different sequence in the middle of sialyl motif S, which is considered to be a splicing variant (Fig. 7A).
一方、他の哺乳動物においてもこれと同様の酵素が存在するのかを調べるため、 ヒ ト ST6Gal IIの配列情報を利用して、 上記と同様にデータベースを検索したと ころ、 マウスにも同様の酵素が存在することが確認できた。 そこでマウスのクロ ーンについてもクローユングを行うことにした。 マウス 14 日目胎児由来 cDNAを 銹型として、 2種類の合成 DNA, 5' -GACAATGGGGATGAGTTTTTTACATCCCAG-3' (図 8 A の 塩 基 番 号 321-350 に 相 当 ） （ 配 列 番 号 1 9 ) , 5' -CGATTTCCTCCCCCAAGGAGGAGTTCAGG-3' (図 8 Aの塩基番号 864-893の相補鎖に相 当） （配列番号 2 0 ) を用いて PCR法により増幅した DNA断片、 および 2種類の 合成 DNA, 5' -ACGTTGGACGGCAGAGAGGCGCCCTTCTCG-3' (図 8 Aの塩基番号 774- 803 に相当） （配列番号 2 1 ) , 5' -ACCTTATTGCACATCAGTTCCCAAGAGTTC-3' (図 8 Aの 塩基番号 1582-1611の相補鎖に相当） （配列番号 2 2 ) を用いて PCR法により増 幅した DNA断片を、両増幅 DNA断片が共通に有する サイ トを利用して連結し、 さらにこれに 2種類の合成 DNA, 5' -CAATGAAACCACACTTGAAGCAATGGCGAC-3' (図 8 A の 塩 基 番 号 1-30 に 相 当 ） （ 配 列 番 号 2 3 ) , 5' -CGCAACAAAAAAATAGCTATCTTCCTCGGG-3' (図 8 Aの塩基番号 381-410の相補鎖に 相当） （配列番号 2 4 ) を用いて PCR法により増幅した DNA断片を、 両 DNA断片 が共通に有する 51HIサイトを利用して連結して、 マウス ST6Gal IIの全長を コードする DNA断片を得、 クローニングベクター pBluescript II SK (+)に挿入し た。 図 8 Aにマウスの ST6Gal IIの配列情報を示す。 マウス ST6Gal IIは 524ァ ミノ酸からなり、 ヒ ト ST6Gal IIより 5アミノ酸ほどステム領域に相当する部分 が短かった。 なお本タンパク質の膜貫通ドメインは、 疎水性分布図によりアミノ 酸番号 12-30 の領域に存在することが予測された （図 8 B ) 。 ヒ トとマウスの ST6Gal IIではァミノ酸配列レベルで 77. 1%の相同性を示した（図 9 B )。 On the other hand, in order to investigate whether or not the same enzyme exists in other mammals, the database was searched in the same manner as above using the sequence information of human ST6Gal II. Was confirmed to exist. Therefore, we decided to clone the mouse clone. Two types of synthetic DNA, 5'-GACAATGGGGATGAGTTTTTTACATCCCAG-3 '(corresponding to base number 321-350 in FIG. 8A), using cDNA derived from mouse fetus day 14 as rust (sequence number 19), 5′-CGATTTCCTCCCCCAAGGAGGAGTTCAGG-3 ′ (corresponding to the complementary strand of base numbers 864-893 in FIG. 8A) (SEQ ID NO: 20), a DNA fragment amplified by PCR and two types of synthetic DNA, 5 ′ -ACGTTGGACGGCAGAGAGGCGCCCTTCTCG-3 '(corresponding to base numbers 774-803 in FIG. 8A) (SEQ ID NO: 21), 5'-ACCTTATTGCACATCAGTTCCCAAGAGTTC-3' (corresponding to the complementary strand of base numbers 1582-1611 in FIG. 8A) No. 22), the DNA fragments amplified by the PCR method were ligated using the site shared by both amplified DNA fragments, and two types of synthetic DNA, 5′-CAATGAAACCACACTTGAAGCAATGGCGAC-3 ′ were further added. (Corresponding to base numbers 1-30 in Fig. 8A) (Sequence number 23), 5'-CGCAACAAAAAAATAGCTATCTTCCTCGGG-3 '(phases in base numbers 381-410 in Fig. 8A) The DNA fragment amplified by PCR using (SEQ ID NO: 24) was ligated using the 51HI site shared by both DNA fragments, and a DNA fragment encoding the full-length mouse ST6Gal II was obtained. The resulting plasmid was inserted into the cloning vector pBluescript II SK (+). FIG. 8A shows the sequence information of mouse ST6Gal II. Mouse ST6Gal II is 524 It was composed of amino acids, and the portion corresponding to the stem region was about 5 amino acids shorter than that of human ST6Gal II. The transmembrane domain of this protein was predicted to be present in the region of amino acid number 12-30 from the hydrophobic distribution diagram (FIG. 8B). Human and mouse ST6Gal II showed 77.1% homology at the amino acid sequence level (FIG. 9B).
つぎに ST6Gal IIの酵素学的諸性質を調べるため、 分泌型タンパク質の製造を 行った。 まずヒ ト ST6Gal II について、 Xhol サイ トを含む合成 DNA, 5， -TCATCTACTTCACCTCGAGCAACCCCGCTG-3' (図 7 Aの塩基番号 255-284に相当） （配 列番号 2 5 ) を用いて膜貫通ドメイン直下流に ol サイ トを導入し、 これと pBluescript II SK (+)由来の ¾oIサイ トを用いて ST6Gal IIのステム領域と活性 ドメインをコードする ¾oI断片を調製した。 これを哺乳動物発現ベクター pcDSA の ¾oIサイ トに挿入した。 この発現べクタ一を pcDSA- hST6Gal I Iと命名した。 またマウス ST6Gal II については、 上記クローユングの際に用いた合成 DNA, 5' -CAATGAAACCACACTTGAAGCAATGGCGAC-3' (図 8 Aの塩基番号 1-30に相当） （配列 番 号 2 3 ) の カゝ わ り に 、 Miwl サ イ ト を 含 む 合 成 DNA, 5' -CATCCAATTGACCAACAGCAATCCTGCGGC-3' (図 8 Aの塩基番号 83-112に相当） （配 列番号 2 6 ) を用いてマウス ST6Gal IIのステム領域と活性ドメインをコードす る MunHhol断片を調製した。これを pcDSAの coRI- J¾oIサイ トに挿入したもの を、 発現ベクター pcDSA- mST6Gal II と命名した。  Next, in order to examine the enzymatic properties of ST6Gal II, secretory proteins were produced. First, for human ST6Gal II, the synthetic DNA containing the Xhol site, 5, -TCATCTACTTCACCTCGAGCAACCCCGCTG-3 '(corresponding to base numbers 255-284 in Fig. 7A) (sequence number 25) was used to directly downstream of the transmembrane domain. An ol site was introduced into the plasmid, and a ¾oI fragment encoding the stem region and active domain of ST6Gal II was prepared using the ol site and a ¾oI site derived from pBluescript II SK (+). This was inserted into the ¾oI site of the mammalian expression vector pcDSA. This expression vector was named pcDSA-hST6GalII. For mouse ST6Gal II, the synthetic DNA used for the clawing described above, 5′-CAATGAAACCACACTTGAAGCAATGGCGAC-3 ′ (corresponding to base numbers 1-30 in FIG. 8A) (SEQ ID NO: 23) Synthetic DNA containing Miwl site, 5'-CATCCAATTGACCAACAGCAATCCTGCGGC-3 '(corresponding to base numbers 83-112 in Fig. 8A) (SEQ ID NO: 26) using mouse ST6Gal II stem region and activity A MunHhol fragment encoding the domain was prepared. This was inserted into the coRI-JoI site of pcDSA and named the expression vector pcDSA-mST6GalII.
pcDSA-hST6Gal IIおよび pcDSA- mST6Gal IIは、 それぞれマウス免疫グロブリ ン IgMのシグナノレぺプチドと Staphylococcus aureus protein A, およびマウス またはヒ ト ST6Gal IIの活性ドメイン（ヒ ト ST6Gal IIではアミノ酸番号 33- 529、 マウス ST6Gal IIではアミノ酸番号 31-524)からなる分泌型融合タンパク質をコ 一ドする。  pcDSA-hST6Gal II and pcDSA-mST6Gal II are the immunoglobulin IgM signapeptide and Staphylococcus aureus protein A, and the active domain of mouse or human ST6Gal II (amino acids 33-529 in human ST6Gal II, respectively). In ST6Gal II, a secretory fusion protein consisting of amino acids 31-524) is encoded.
各発現ベクターとリポフエク トァミン（Invitrogen)を用いて C0S-7細胞でその 一過性発現を行った（Koj ima, N. et a J. (1995) FEES Lett. 360, 1-4)。 ここで それぞれの発現べクターを導入した細胞から細胞外に分泌された本発明のタンパ ク質を PA-hST6Gal Π (ヒ ト）および PA- mST6Gal II (マウス）と命名した。 PA-hST6Gal II、 PA- mST6Gal IIは IgG- Sepharose (Amersham Pharmacia Biotech 社）に吸着させて培地より回収した。シアル酸転移酵素活性は Leeらの方法に準じ て以下のよ う に行った（Lee, Y. - C. et al. (1999) J. Biol. Chem. 274, 11958—11967)。 50 mM MESバッファー（pH 6. 0) , 1 mM MgCl2, 1 raM CaCl2, 0. 5% Triton CF-54, 100 μ Μ CMP- [14C] - NeuAc, 基質糖鎖（糖脂質の場合は 0. 5 rag/ml， 糖タン パク質、 オリゴ糖は 1 mg/mlになるように添加） 、 および PA- hST6Gal IIまたは PA-raST6Gal II懸濁液を含む反応液（10 1)を 37度で 3-20時間ィンキュベート し、 その後、 糖脂質については C- 18カラム（Sep- Pak Vac 100 mg ; Waters社）を 用いて精製したものを試料として、 オリゴ糖、 糖タンパク質については反応産物 をそのまま試料として解析を行った。 オリゴ糖、 糖脂質はシリカゲル 60HPTLCプ レート（Merck社）にスポットし、 1-プロパノール：アンモニア水：水 =6 : 1 : 2. 5の 展開溶媒 （オリゴ糖用）またはクロ口ホルム： メタノール： 0. 02% CaCl2=55 : 45 : 10 の展開溶媒 （糖脂質用） で展開した。 糖タンパク質の場合は SDS-ポリアクリルァ ミ ドゲル電気泳動によって解析を行った。 これらの放射活性を BAS2000ラジオィ メージアナライザー （フジフィルム） で可視化し、 定量した。 Using each expression vector and Lipofectamine (Invitrogen), the cells were transiently expressed in COS-7 cells (Kojima, N. et a J. (1995) FEES Lett. 360, 1-4). Here, the proteins of the present invention secreted extracellularly from the cells into which the respective expression vectors were introduced were named PA-hST6Gal II (human) and PA-mST6Gal II (mouse). PA-hST6Gal II and PA-mST6Gal II were adsorbed on IgG-Sepharose (Amersham Pharmacia Biotech) and recovered from the medium. Sialyltransferase activity was performed as follows according to the method of Lee et al. (Lee, Y.-C. et al. (1999) J. Biol. Chem. 274, 11958-11967). 50 mM MES buffer (pH 6.0), 1 mM MgCl 2 , 1 raM CaCl 2 , 0.5% Triton CF-54, 100 μΜ CMP- [ 14 C]-NeuAc, substrate sugar chain (for glycolipids) Add 0.5 rag / ml, add glycoprotein and oligosaccharide to 1 mg / ml), and add a reaction solution (101) containing PA-hST6Gal II or PA-raST6Gal II suspension. After incubating for 3-20 hours at room temperature, the glycolipid was purified using a C-18 column (Sep-Pak Vac 100 mg; Waters), and the reaction product was analyzed for oligosaccharides and glycoproteins. The analysis was performed as a sample as it was. Oligosaccharides and glycolipids are spotted on silica gel 60HP TLC plate (Merck) and developed with 1-propanol: ammonia water: water = 6: 1: 2.5 developing solvent (for oligosaccharides) or chloroform: methanol: 0. It was developed with a developing solvent (for glycolipids) of 02% CaCl 2 = 55: 45: 10. For glycoproteins, analysis was performed by SDS-polyacrylamide gel electrophoresis. The radioactivity was visualized and quantified using a BAS2000 radio image analyzer (Fujifilm).
表 2に PA_hST6Gal Π、 PA- mST6Gal IIの基質特異性を示す。 Table 2 shows the substrate specificity of PA_hST6GalII and PA-mST6GalII.
表 2 Table 2
ST6Gal IIの基質特異性  ST6Gal II substrate specificity
PA-hST6Gal IIおよび PA-mST6Gal II を用いて様々な基質に対する特異性を検討した。 各基質の濃度は、 糖脂質の塌合は 0.5 mg/mlに、 糖タンパク質、 単糖、 オリゴ糖の場合は 1 mg/mlになるようにした。 相対活性は Galpl,4GlcNAcの取り込み値を 100として計算した。 Rは W型糖鎖の残りの糖鎖部分を意味する。 PA-hST6Gal II and PA-mST6Gal II were used to examine their specificity for various substrates. The concentration of each substrate was 0.5 mg / ml for glycolipids and 1 mg / ml for glycoproteins, monosaccharides and oligosaccharides. The relative activity was calculated with the incorporation value of Galpl, 4GlcNAc as 100. R means the remaining sugar chain part of the W-type sugar chain.
Acceptors Representative structures of carbohydrates ciativc rate Acceptors Representative structures of carbohydrates ciativc rate
Mouse Human Human  Mouse Human Human
STWal II ST6Gal 1 STWal II ST6Gal 1
Figure imgf000040_0001
Figure imgf000040_0001
Type I GaJpl^GlcNAc 0 0 4.2  Type I GaJpl ^ GlcNAc 0 0 4.2
Type ΙΠ Gaipi,3GalNAc 0 0 0  Type ΙΠ Gaipi, 3GalNAc 0 0 0
fi al ft 1 4Glc o o 8 7  fi al ft 1 4Glc o o 8 7
Lacto-W-tetraose Gal 1 ,3GlcNAcp 1 ,3Ga^l ,4Glc o o 31.1  Lacto-W-tetraose Gal 1, 3GlcNAcp 1, 3Ga ^ l, 4Glc o o 31.1
一  One
n π  n π
一 one
3 a 3a
o o n  o o n
i  i
Asialo-BSM 0 0 0  Asialo-BSM 0 0 0
Ovomucoid NeuAca2,3Gaip l,4GlcNAc-R 0 0 9.0  Ovomucoid NeuAca2,3Gaip l, 4GlcNAc-R 0 0 9.0
Figure imgf000040_0002
Figure imgf000040_0002
* 2.74 pmol/h/ml medium. **, 1.03 pmol/h/ml medium. *** 8.14 pmol/h/ml medium. NeuAc, -acetylneuraminic acid. Cer, ceiamide. * 2.74 pmol / h / ml medium. **, 1.03 pmol / h / ml medium. *** 8.14 pmol / h / ml medium. NeuAc, -acetylneuraminic acid.Cer, ceiamide.
両酵素ともオリゴ糖に対しては、非還元末端に Gal β 1, 4GlcNAc構造をもつもの に対してのみ活性を示した（図 1 0 )。 またこの構造を持つと考えられる糖タンパ ク質に対しても弱い活性を示した。 一方、 糖脂質については、 調べた範囲内では 両酵素の基質となるものはなかった。 また比較のためにヒ ト ST6Gal Iのオリゴ糖 に対する活性を調べたところ、 Gal ]3 1, 4GlcNAc 構造をもつオリゴ糖のほかに、 Lactoseや Lacto- -tetraoseなどに対しても活性を示した（図 1 0 )。また ST6Gal I は糖タンパク質や糖脂質に対しても広い活性を示した（表 2 )。 以上のことは ST6Gal IIが ST6Gal Iよりも基質特異性に関してより選択性が強いことを意味す る。 なおヒ ト ST6Gal IIの splicing variantである short formのタンパク質に ついては、 酵素活性が認められなかった（図 1 0 )。 Both enzymes showed activity against oligosaccharides only on those with Galβ1,4GlcNAc structure at the non-reducing end (Fig. 10). It also showed weak activity against glycoproteins that are thought to have this structure. On the other hand, no glycolipid was a substrate for both enzymes within the range examined. For comparison, the activity of human ST6Gal I on oligosaccharides was examined. In addition to the oligosaccharides having the Gal] 31,4GlcNAc structure, they also showed activity on Lactose, Lacto-tetraose, etc. ( (Figure 10). ST6Gal I also showed broad activity on glycoproteins and glycolipids (Table 2). This implies that ST6Gal II is more selective in substrate specificity than ST6Gal I. No enzymatic activity was observed for the short form of the protein, a splicing variant of human ST6Gal II (Fig. 10).
PA-hST6Gal IIおよび PA- mST6Gal IIにより Gal β 1, 4GlcNAcにシアル酸を転移 した場合、その反応産物の導入シアル酸は ST6Gal Iの場合と同様に ct 2, 3-結合で 結合しているシアル酸を特異的に切断するシァリダーゼ（NA ase I)では切断され なかったが、 α 2, 3-, α 2， 6-結合で結合しているシアル酸を特異的に切断するシ ァリダーゼ（NANase II)では切断された（図 1 1 A)。またこの反応産物は TLCにお いて 6， - sialyl- -acetyllactosamine と同じ移動度を示したこと、 さらにガラク トシダーゼ処理では TLC において移動度に変化が認められなかったことから（図 1 1 Β )、 α 2, 6 結合を介してガラク トースにシアル酸が導入された 6'- sialyl- - acetyllactosamineであると考えられた。 以上により ST6Gal II は シアル酸を α 2, 6-の結合様式でガラク トースに転移することが明らかになった。 なおその特に好ましい基質としては、非還元末端に Gal j3 1, 4GlCNAc構造をもつォ リゴ糖と考えられた。 When sialic acid is transferred to Galβ1,4GlcNAc by PA-hST6Gal II and PA-mST6Gal II, the sialic acid introduced into the reaction product is sialic acid bound by ct2,3-linkage as in ST6GalI. A sialidase that specifically cleaved sialic acid (NANase II) was not cleaved by sialidase that specifically cleaves acid (NAase I), but specifically cleaves sialic acid bound by α2,3-, α2,6-linkage. ) Was cut (Fig. 11A). In addition, this reaction product showed the same mobility as that of 6, -sialyl- -acetyllactosamine in TLC, and no change was observed in the mobility of TLC after galactosidase treatment (Fig. 11Β). It was considered to be 6'-sialyl-acetylacetyltosamine in which sialic acid was introduced into galactose via α 2,6 bond. From the above, it was clarified that ST6Gal II transfers sialic acid to galactose in the α2,6-linkage mode. A particularly preferred substrate was considered to be an oligosaccharide having a Gal j31,4G1 C NAc structure at the non-reducing end.
またヒ ト ST6Gal I, ST6Gal IIの様々な組織における発現パターンを、 ST6Gal I 特異的プライマー （ 5'_TTATGATTCACACCAACCTGAAG- 3' (配列番号 2 7 ) および 5'-CTTTGTACTTGTTCATGCTTAGG-3' (配列番号 2 8 )、PCR増幅断片の大きさは 372 bp) と ST6Gal II 特異的プライマー （5， - AGACGTCATTTTGGTGGCCTGGG- 3， （図 7 Aの塩 基番号 1264-1286に相当） （配列番号 2 9 ) および 5'- TTAAGAGTGTGGMTGACTGG - 3' (図 7 Aの塩基番号 1745- 1765に相当） （配列番号 3 0 ) 、 PCR増幅断片の大きさ は 502 bp) を用いて PCR法で調べた （図 1 2 A) 。 ヒ ト ST6Gal Iはほとんどの 組織で発現していたが、 ST6Gal II は小腸、 大腸、 胎児脳を除く組織での発現は 非常に低いか、 全く認められなかった。 さらにヒ ト ST6Gal Iは各種腫瘍細胞で発 現していたが、 ST6Gal II の発現は検出できなかった（図 1 2 B )。 またマウス ST6Gal II の発現様式について、 マウス ST6Gal II 特異的プライマー (5' -CAATGAAACCACACTTGAAGCAATGGCGAC-3' (図 8 Aの塩基番号 1-30に相当） （配列 番号 2 3 ) および 5' -CGCAACAAAAAAATAGCTATCTTCCTCGGG-3' (図 8 Aの塩基番号 381- 410の相補鎖に相当） （配列番号 2 4 ) 、 PCR増幅断片の大きさは 410 bp)を 用いて同様に調べたところ、 脳および胎生期でその発現が認められたが、 その他 の組織での発現は非常に低い力、 全く認められなかった （図 1 2 C ) 。 以上の結 果は ST6Gal Iと ST6Gal IIが生体内で異なる役割を果たしていることを示唆する。 産業上の利用の可能性 The expression patterns of human ST6Gal I and ST6Gal II in various tissues were determined using ST6Gal I-specific primers (5′_TTATGATTCACACCAACCTGAAG-3 ′ (SEQ ID NO: 27) and 5′-CTTTGTACTTGTTCATGCTTAGG-3 ′ (SEQ ID NO: 28), The size of the PCR amplified fragment is 372 bp) and ST6Gal II specific primers (5, -AGACGTCATTTTGGTGGCCTGGG-3, (corresponding to base number 1264-1286 in Fig. 7A) (SEQ ID NO: 29)) and 5'-TTAAGAGTGTGGMTGACTGG- 3 ' (Corresponding to base numbers 1745 to 1765 in FIG. 7A) (SEQ ID NO: 30), and the size of the PCR amplified fragment was determined by PCR using 502 bp (FIG. 12A). Human ST6Gal I was expressed in most tissues, but ST6Gal II was very low or not expressed in tissues other than the small intestine, large intestine, and fetal brain. Furthermore, human ST6Gal I was expressed in various tumor cells, but ST6Gal II expression could not be detected (Fig. 12B). Regarding the expression pattern of mouse ST6Gal II, the mouse ST6Gal II-specific primers (5'-CAATGAAACCACACTTGAAGCAATGGCGAC-3 '(corresponding to nucleotide numbers 1-30 in FIG. 8A) (SEQ ID NO: 23) and 5'-CGCAACAAAAAAATAGCTATCTTCCTCGGG-3' (Corresponding to the complementary strand of base numbers 381-410 in Fig. 8A) (SEQ ID NO: 24). The size of the PCR amplified fragment was similarly examined using However, expression in other tissues was very low and was not observed at all (Fig. 12C). The above results suggest that ST6Gal I and ST6Gal II play different roles in vivo. Industrial applicability
本発明により新規酵素として O- glycan a 2, 8-シアル酸転移酵素、および該酵素 の活性部分を有し細胞外に分泌される新規蛋白質が提供される。 本発明の酵素お よび蛋白質は、 0- glycan a 2, 8-シアル酸転移酵素活性を有するので、 例えば、 蛋 白にヒ ト型の糖鎖を導入する試薬として有用である。 また、 本発明の O - glycan α 2, 8-シアル酸転移酵素は、 ヒ トに特異的な糖鎖を欠く遺伝性疾患の治療のため の医薬として有用である。さらに、本発明の Ο - glycan ο; 2, 8-シアル酸転移酵素は、 癌転移抑制、 ウィルス感染防止、 炎症反応抑制、 神経組織賦活作用を目的とする 医薬としても用いることが可能である。 さらにまた、 本発明の Ο - glycan α 2, 8- シアル酸転移酵素は、 薬剤等にシアル酸を付加することにより生理作用を増加さ せるための研究用試薬などとして有用である。 The present invention O- glyca na 2 as a new enzyme, 8-sialyltransferase and novel protein that is secreted outside of the cell has an active portion of the enzyme is provided by. Since the enzyme and protein of the present invention have 0-glycana 2,8-sialyltransferase activity, they are useful, for example, as reagents for introducing human sugar chains into proteins. Also, O of the present invention - glycan α 2, 8- sialyltransferase is useful as a medicament for the treatment of genetic disorders that lack specific sugar chain human. Furthermore, the glycan o; 2,8-sialyltransferase of the present invention can also be used as a medicament for the purpose of suppressing cancer metastasis, preventing virus infection, suppressing inflammatory response, and activating nerve tissue. Furthermore, the Ο-glycan α2,8-sialyltransferase of the present invention is useful as a research reagent or the like for increasing a physiological action by adding sialic acid to a drug or the like.
さらに本発明により新規酵素として β—ガラク トシド α 2 , 6—シアル酸転移 酵素、 および該酵素の活性部分を有し細胞外に分泌される新規蛋白質が提供され る。 本発明の酵素および蛋白質は /3—ガラク トシド α 2， 6—シアル酸転移酵素 活性を有するので、 Gal/31,4GlcNAc構造をもつオリゴ糖などのガラク トース上に α2,6 の結合様式でシアル酸をより選択的に導入することが可能になった。 本発 明の /3—ガラクトシド α2,6-シアル酸転移酵素 ST6Gal IIは、 本酵素が合成する 特異的な糖鎖を欠く遺伝性疾患の治療薬として、 また癌転移抑制、 ウィルス感染 抑防止、 炎症反応抑制、 神経細胞賦活効果を有する薬剤として、 あるいは糖鎖に シアル酸を付加することにより生理作用を増加させたり、 糖鎖分解酵素の分解活 性を阻害する研究用試薬などとして有用である。 Further, the present invention provides β-galactoside α2,6-sialyltransferase as a novel enzyme and a novel protein having an active portion of the enzyme and secreted extracellularly. The enzyme and the protein of the present invention comprise a 3 / 3-galactoside α2,6-sialyltransferase Due to its activity, it became possible to introduce sialic acid more selectively into a galactose such as an oligosaccharide having a Gal / 31,4GlcNAc structure in an α2,6 binding mode. The / 3-galactoside α2,6-sialyltransferase ST6Gal II of the present invention is a therapeutic drug for hereditary diseases lacking the specific sugar chain synthesized by this enzyme, and also suppresses cancer metastasis, virus infection, It is useful as a drug that has an inflammatory response inhibitory or neuronal activation effect, or as a research reagent that increases the physiological action by adding sialic acid to sugar chains, or inhibits the degradation activity of glycolytic enzymes. .

Claims

請求の範囲 The scope of the claims
1. 以下の基質特異性および基質選択性を有することを特徴とする、 o1. characterized by having the following substrate specificity and substrate selectivity, o
-glycan a 2, 8-シアル酸転移酵素。 -glycan a 2, 8-sialyltransferase.
基質特異性：末端に Siaa2,3(6)Gal (ここで、 Siaはシアル酸を示し、 Galは ガラクトースを示す)構造をもつ糖を基質とする ；  Substrate specificity: a sugar having a terminal structure of Siaa2,3 (6) Gal (where Sia represents sialic acid and Gal represents galactose) is used as a substrate;
基質選択性：糖脂質および N型糖鎖よりも優先的に O型糖鎖に対してシアル酸 を取り込ませる ：  Substrate selectivity: Incorporates sialic acid into O-glycans preferentially over glycolipids and N-glycans:
2. 下記の何れかのアミノ酸配列を有する 0- glycan α2，8-シアル酸転移酵  2. 0-glycan α2,8-sialyltransferase having any of the following amino acid sequences:
( 1 ) 配列表の配列番号 1または 3に記載のァミノ酸配列；又は (1) an amino acid sequence according to SEQ ID NO: 1 or 3 in the sequence listing; or
( 2 ) 配列表の配列番号 1または 3に記載のァミノ酸配列において 1から数個の アミノ酸の欠失、置換及びノ又は付加を有するアミノ酸配列を有し、 Ο - glycan α (2) an amino acid sequence described in SEQ ID NO: 1 or 3 in the sequence listing, which has an amino acid sequence having deletion, substitution and no or addition of one to several amino acids, and Ο-glycan α
2, 8-シアル酸転移を触媒する活性を有するァミノ酸配列： Amino acid sequence that has the activity of catalyzing 2, 8-sialyl transfer:
3. 請求項 2に記載の O-glycan α 2， 8-シアル酸転移酵素のアミノ酸配列を コードする〇- glycan α 2, 8-シアル酸転移酵素遺伝子。  3. A 〇-glycan α2,8-sialyltransferase gene encoding the amino acid sequence of the O-glycan α2,8-sialyltransferase according to claim 2.
4. 下記の何れかの塩基配列を有する請求項 3に記載の Ο- glycan α2,8-シ アル酸転移酵素。  4. The Ο-glycan α2,8-sialyltransferase according to claim 3, having any one of the following nucleotide sequences.
(1) 配列表の配列番号 2に記載の塩基配列中の塩基番号 7 7番目から 1 2 70 番目で特定される塩基配列；  (1) a nucleotide sequence identified by nucleotide numbers 77 to 127 in the nucleotide sequence of SEQ ID NO: 2 in the sequence listing;
(2) 配列表の配列番号 2に記載の塩基配列中の塩基番号 7 7番目から 1 2 70 番目で特定される塩基配列において 1から数個の塩基の欠失、 置換及びノ又は付 加を有する塩基配列を有し、 Ο - gl y can α 2, 8-シァル酸転移を触媒する活性を有 する蛋白質をコードする塩基配列：  (2) In the nucleotide sequence of nucleotides 77 to 127 in the nucleotide sequence of SEQ ID NO: 2 in the sequence listing, deletion, substitution, and addition or deletion of one to several nucleotides are included. A nucleotide sequence that encodes a protein having an activity of catalyzing 転 移 -glycan α2,8-sialyltransferase:
(3) 配列表の配列番号 4に記載の塩基配列中の塩基番号 9 2番目から 1 28 5 番目で特定される塩基配列；  (3) a base sequence specified by base numbers 9 from 2 to 1285 in the base sequence of SEQ ID NO: 4 in the sequence listing;
(4) 配列表の配列番号 4に記載の塩基配列中の塩基番号 9 2番目から 1 28 5 番目で特定される塩基配列において 1から数個の塩基の欠失、 置換及び 又は付 加を有する塩基配列を有し、 0- glycan ct 2, 8-シアル酸転移を触媒する活性を有 する蛋白質をコードする塩基配列： (4) Nucleotide number 9 in the nucleotide sequence described in SEQ ID NO: 4 in the sequence listing from the second to 1 285 A protein having an activity of catalyzing the transfer of 0-glycan ct 2, 8-sialic acid, having a base sequence having deletion, substitution, and / or addition of one to several bases in the base sequence specified by Nucleotide sequence encoding:
5 . 請求項 3または 4に記載の 0- glycan α 2, 8-シアル酸転移酵素遺伝子を 含む組み換えベクター。  5. A recombinant vector comprising the 0-glycan α2,8-sialyltransferase gene according to claim 3 or 4.
6 . 発現ベクターである、 請求項 5に記載の組み換えベクター。  6. The recombinant vector according to claim 5, which is an expression vector.
7 . 請求項 5または 6に記載の組み換えベクターにより形質転換された形質 転換体。  7. A transformant transformed with the recombinant vector according to claim 5 or 6.
8 . 請求項 7に記載の形質転換体を培養し培養物から請求項 1または 2に記 載の酵素を採取することを特徴とする、 請求項 1または 2に記載の酵素の製造方 法。  8. The method for producing an enzyme according to claim 1 or 2, wherein the transformant according to claim 7 is cultured, and the enzyme according to claim 1 or 2 is collected from the culture.
9 . 下記の何れかのアミノ酸配列を有する 0- glycan α 2, 8-シアル酸転移酵 素活性ドメインから成る蛋白質。  9. A protein comprising a 0-glycan α2,8-sialyltransferase active domain having any one of the following amino acid sequences:
( 1 ) 配列表の配列番号 1に記載のァミノ酸配列のァミノ酸番号 2 6〜 3 9 8力 ら成るアミノ酸配列；  (1) an amino acid sequence consisting of amino acid numbers 26 to 398 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing;
( 2 ) 配列表の配列番号 1に記載のァミノ酸配列のァミノ酸番号 2 6〜 3 9 8か ら成るアミノ酸配列において 1から数個のアミノ酸の欠失、 置換及び Ζ又は付加 を有するアミノ酸配列を有し、 O-glycan α 2, 8-シアル酸転移を触媒する活性を 有するアミノ酸配列：  (2) an amino acid sequence comprising the amino acid sequence of amino acids 26 to 398 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, the amino acid sequence having deletion, substitution and 及 び or addition of one to several amino acids; An amino acid sequence having the activity of catalyzing O-glycan α2, 8-sialyltransferase:
( 3 ) 配列表の配列番号 3に記載のァミノ酸配列のァミノ酸番号 6 8〜 3 9 8か ら成るアミノ酸配列；又は  (3) an amino acid sequence consisting of amino acid numbers 68 to 398 of the amino acid sequence described in SEQ ID NO: 3 in the sequence listing; or
( 4 ) 配列表の配列番号 3に記載のァミノ酸配列のァミノ酸番号 6 8〜 3 9 8か ら成るアミノ酸配列において 1から数個のァミノ酸の欠失、 置換及びノ又は付加 を有するアミノ酸配列を有し、 0- glycan α 2, 8-シアル酸転移を触媒する活性を 有するアミノ酸配列：  (4) an amino acid having one to several amino acid deletions, substitutions and no or additions in the amino acid sequence consisting of amino acid numbers 68 to 398 of the amino acid sequence described in SEQ ID NO: 3 in the sequence listing An amino acid sequence having an activity of catalyzing 0-glycan α 2, 8-sialyltransferase:
1 0 . 請求項 1または 2に記載の Ο- glycan α 2, 8-シアル酸転移酵素の活性 ドメィンであるポリぺプチド部分とシグナルぺプチドとを含む細胞外分泌型の蛋 白であって、 O- glycan α2， 8 -シアル酸転移を触媒する活性を有する蛋白質。10. The activity of the Ο-glycan α2,8-sialyltransferase according to claim 1 or 2, and an extracellular secretory protein comprising a polypeptide moiety that is a domain and a signal peptide. A white protein that has the activity of catalyzing O-glycan α 2,8-sialyltransferase.
1 1. 請求項 9又は 1 0に記載の蛋白質をコードする遺伝子。 1 1. A gene encoding the protein according to claim 9 or 10.
1 2. 請求項 1 1に記載の遺伝子を含む組み換えベクター。  1 2. A recombinant vector containing the gene according to claim 11.
1 3. 発現ベクターである、 請求項 1 2に記載の組み換えベクター。  1 3. The recombinant vector according to claim 12, which is an expression vector.
1 4. 請求項 1 2または 1 3に記載の組み換えベクターにより形質転換され た形質転換体。  1 4. A transformant transformed with the recombinant vector according to claim 12 or 13.
1 5. 請求項 1 4に記載の形質転換体を培養し培養物から請求項 9または 1 0に記載の蛋白質を採取することを特徴とする、 請求項 9または 1 0に記載の蛋 白質の製造方法。  1 5. The method according to claim 9 or 10, wherein the transformant according to claim 14 is cultured, and the protein according to claim 9 or 10 is collected from the culture. Production method.
1 6. 以下の作用および基質特異性を有することを特徴とする、 一ガラク トシド α 2 , 6—シアル酸転移酵素。  1 6. A galactoside α2,6-sialyltransferase, which has the following action and substrate specificity.
( 1 ) 作用 ；  (1) action;
末端にガラク トース ] 3 1 , 4 Ν—ァセチルダルコサミン構造をもつ糖鎖のガラ ク トース部分に α 2， 6の結合様式でシアル酸を転移する。  Galactose at the terminus] 31 Sialic acid is transferred to the galactose portion of the sugar chain having a 3,4'-acetyldarcosamine structure by α2,6 linkage.
( 2 ) 基質特異性；  (2) substrate specificity;
末端にガラク トース / 3 1， 4 Ν—ァセチルダルコサミン構造をもつ糖鎖を基質 とし、 ラク トース、 及び末端にガラク トース β 1 , 3 N—ァセチルダルコサミン 構造をもつ糖鎖を基質としない。  A sugar chain having a galactose / 31,4'-acetyldarcosamine structure at the end is used as a substrate, and a lactose and a sugar chain having a galactose β1,3N-acetyldarcosamine structure at the end are used as substrates. do not do.
1 7. 下記の何れかのアミノ酸配列を有する ]3—ガラク トシド 2, 6—シ アル酸転移酵素。  1 7. Having any one of the following amino acid sequences: 3-galactoside 2,6-sialyltransferase.
( 1 ) 配列表の配列番号 5または 7に記載のァミノ酸配列；又は  (1) an amino acid sequence according to SEQ ID NO: 5 or 7 in the sequence listing; or
( 2 ) 配列表の配列番号 5または 7に記載のァミノ酸配列において 1から数個の アミノ酸の欠失、 置換及び/又は付加を有するアミノ酸配列を有し、 β—ガラク トシド 2， 6 _シアル酸転移を触媒する活性を有するアミノ酸配列：  (2) a β-galactoside 2,6-sial having an amino acid sequence having deletion, substitution and / or addition of one to several amino acids in the amino acid sequence described in SEQ ID NO: 5 or 7 in the sequence listing; Amino acid sequence having activity for catalyzing acid transfer:
1 8. 請求項 1 7に記載の /3—ガラク トシド α 2 , 6—シアル酸転移酵素の ァミノ酸配列をコードする ]3—ガラク トシド α 2， 6 _シアル酸転移酵素遺伝子。  1 8. The 3-galactoside α2,6_sialyltransferase gene encoding the amino acid sequence of the / 3-galactoside α2,6-sialyltransferase according to claim 17.
1 9. 下記の何れかの塩基配列を有する請求項 1 8に記載の 3—ガラク トシ ド α 2 , 6—シアル酸転移酵素遺伝子。 19. The 3-galactosidase according to claim 18, which has any one of the following nucleotide sequences: De α 2, 6-Sialyltransferase gene.
(1) 配列表の配列番号 6に記載の塩基配列中の塩基番号 1 76番目から 1 76 2番目で特定される塩基配列；  (1) a base sequence specified by base numbers 176 to 1762 in the base sequence described in SEQ ID NO: 6 in the sequence listing;
(2) 配列表の配列番号 6に記載の塩基配列中の塩基番号 1 76番目から 1 76 2番目で特定される塩基配列において 1から数個の塩基の欠失、 置換及び/又は 付加を有する塩基配列を有し、 ]3—ガラク トシド α 2， 6—シアル酸転移を触媒 する活性を有する蛋白質をコードする塩基配列：  (2) In the nucleotide sequence represented by nucleotide numbers 176 to 176 in the nucleotide sequence described in SEQ ID NO: 6 in the sequence listing, the nucleotide sequence has deletion, substitution, and / or addition of one to several nucleotides. A nucleotide sequence encoding a protein having a nucleotide sequence and having an activity of catalyzing 3-galactoside α2,6-sialyltransferase:
(3) 配列表の配列番号 8に記載の塩基配列中の塩基番号 3番目から 1 5 74番 目で特定される塩基配列；又は  (3) a nucleotide sequence specified by nucleotides 3 through 1574 in the nucleotide sequence of SEQ ID NO: 8 in the sequence listing; or
(4) 配列表の配列番号 8に記載の塩基配列中の塩基番号 3番目から 1 5 74番 目で特定される塩基配列において 1から数個の塩基の欠失、 置換及び Ζ又は付加 を有する塩基配列を有し、 i3 _ガラク トシド α 2, 6—シアル酸転移を触媒する 活性を有する蛋白質をコードする塩基配列：  (4) In the nucleotide sequence identified by nucleotide numbers 3 to 1574 in the nucleotide sequence described in SEQ ID NO: 8 in the sequence listing, the nucleotide sequence has deletion, substitution, 及 び or addition of one to several nucleotides. A nucleotide sequence encoding a protein having a nucleotide sequence and having an activity of catalyzing i3_galactoside α2,6-sialyltransferase:
20. 請求項 1 8または 1 9に記載の 一ガラク トシド α 2, 6—シアル酸 転移酵素遺伝子を含む組み換えベクター。  20. A recombinant vector comprising the monogalactoside α2,6-sialyltransferase gene according to claim 18 or 19.
2 1. 発現ベクターである、 請求項 20に記載の組み換えベクター。  21. The recombinant vector according to claim 20, which is an expression vector.
2 2. 請求項 20または 2 1に記載の組み換えベクターにより形質転換され た形質転換体。  2 2. A transformant transformed with the recombinant vector according to claim 20 or 21.
2 3. 請求項 2 2に記載の形質転換体を培養し培養物から請求項 1 6または 1 7に記載の酵素を採取することを特徴とする、 請求項 1 6たは 1 7に記載の酵 素の製造方法。  2 3. The method according to claim 16, wherein the transformant according to claim 22 is cultured and the enzyme according to claim 16 or 17 is collected from the culture. A method for producing enzymes.
24. 下記の何れかのアミノ酸配列を有する i3—ガラクトシド α 2, 6—シ アル酸転移酵素活性ドメインから成る蛋白質。  24. A protein comprising an i3-galactoside α 2,6-sialyltransferase active domain having any one of the following amino acid sequences:
( 1 ) 配列表の配列番号 5に記載のァミノ酸配列のァミノ酸番号 3 3〜 5 2 9力 ら成るアミノ酸配列；  (1) an amino acid sequence consisting of amino acid numbers 33 to 529 of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing;
( 2 ) 配列表の配列番号 5に記載のァミノ酸配列のァミノ酸番号 33〜 5 2 9力 ら成るアミノ酸配列において 1から数個のアミノ酸の欠失、 置換及び 又は付加 を有するァミノ酸配列を有し、 ]3—ガラクトシド α 2 , 6—シアル酸転移を触媒 する活性を有するアミノ酸配列： (2) Deletion, substitution and / or addition of one to several amino acids in an amino acid sequence consisting of amino acids 33 to 529 of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing An amino acid sequence having an amino acid sequence having the formula: and an activity of catalyzing 3-galactoside α 2, 6-sialyl transfer:
( 3 ) 配列表の配列番号 7に記載のァミノ酸配列のァミノ酸番号 3 1〜 5 2 4か ら成るアミノ酸配列；又は  (3) an amino acid sequence consisting of amino acid numbers 31 to 524 of the amino acid sequence described in SEQ ID NO: 7 in the sequence listing; or
( 4 ) 配列表の配列番号 7に記載のァミノ酸配列のァミノ酸番号 3 1〜 5 2 4か ら成るアミノ酸配列において 1から数個のァミノ酸の欠失、 置換及び Ζ又は付加 を有するアミノ酸配列を有し、 3—ガラクトシド α 2 , 6—シアル酸転移を触媒 する活性を有するアミノ酸配列：  (4) an amino acid having 1 to several amino acid deletions, substitutions and Ζ or additions in an amino acid sequence consisting of amino acid numbers 31 to 524 of the amino acid sequence described in SEQ ID NO: 7 in the sequence listing An amino acid sequence having the sequence and having an activity of catalyzing 3-galactoside α 2, 6-sialyl transfer:
2 5 . 請求項 1 6または 1 7に記載の J3—ガラクトシド α 2， 6—シアル酸 転移酵素の活性ドメィンであるポリぺプチド部分とシグナルぺプチドとを含む細 胞外分泌型の蛋白であって、 /3 _ガラク トシド α 2， 6—シアル酸転移を触媒す る活性を有する蛋白質。  25. An extracellular secretory protein comprising a polypeptide moiety which is an active domain of J3-galactoside α2,6-sialyltransferase according to claim 16 or 17, and a signal peptide. , / 3_galactoside A protein having an activity of catalyzing α2,6-sialyltransferase.
2 6 . 請求項 2 4又は 2 5に記載の蛋白質をコードする遺伝子。  26. A gene encoding the protein according to claim 24 or 25.
2 7 . 請求項 2 6に記載の遺伝子を含む組み換えべクタ一。  27. A recombinant vector containing the gene according to claim 26.
2 8 . 発現ベクターである、 請求項 2 7に記載の組み換えベクター。  28. The recombinant vector according to claim 27, which is an expression vector.
2 9 . 請求項 2 7または 2 8に記載の組み換えベクターにより形質転換され た形質転換体。  29. A transformant transformed with the recombinant vector according to claim 27 or 28.
3 0 . 請求項 2 9に記載の形質転換体を培養し培養物から請求項 2 4または 2 5に記載の蛋白質を採取することを特徴とする、 請求項 2 4または 2 5に記載 の蛋白質の製造方法。  30. The protein according to claim 24 or 25, wherein the transformant according to claim 29 is cultured and the protein according to claim 24 or 25 is collected from the culture. Manufacturing method.
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